Novel delta-endotoxin gene isolated from Bacillus thuringiensis var. finitimus

ABSTRACT

Novel delta-endotoxin genes cry26Aa1 and cry28Aa1 isolated from  Bacillus thuringiensis  ssp.  finitimus,  whose expression results in novel delta-endotoxins, are disclosed herein. The invention also discloses compositions and formulations containing the toxins that are capable of controlling insect pests. The invention is further drawn to methods of making the toxins and to methods of using the genes, for example in microorganisms to control insect pests or in transgenic plants to confer insect resistance.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/175,158, filed Jan. 7, 2000, incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

[0002] The invention relates to novel delta-endotoxin gene familiescry26 and cry28 from Bacillus thuringiensis ssp. finitimus, thedelta-endotoxins resulting from expression of said genes, and methods ofusing the genes and corresponding toxins to control insects.

BACKGROUND OF THE INVENTION

[0003] Insect pests are a major cause of crop losses. Solely in the US,about $7.7 billion are lost every year due to infestation by variousgenera of insects. In addition to losses in field crops, insect pestsare also a burden to vegetable and fruit growers, to producers ofornamental flowers, and they are a nuisance to gardeners and homeowners.

[0004] Insect pests are mainly controlled by intensive applications ofchemical insecticides, which are active through inhibition of insectgrowth, prevention of insect feeding or reproduction, or death of theinsects. Good insect control can thus be reached, but these chemicalscan sometimes also affect other, beneficial insects. Another problemresulting from the wide use of chemical pesticides is the appearance ofresistant insect varieties. This has been partially alleviated byvarious resistance management strategies, but there is an increasingneed for alternative pest control agents. Biological insect controlagents, such as Bacillus thuringiensis strains expressing insecticidaltoxins like δ-endotoxins, have also been applied with satisfactoryresults, offering an alternative or a complement to chemicalinsecticides. Recently, the genes coding for some of these δ-endotoxinshave been isolated and their expression in heterologous hosts have beenshown to provide another tool for the control of economically importantinsect pests. In particular, the expression of insecticidal toxins intransgenic plants, such as Bacillus thuringiensis δ-endotoxins, hasprovided efficient protection against selected insect pests, andtransgenic plants expressing such toxins have been commercialized,allowing farmers to reduce applications of chemical insect controlagents. Yet, even in this case, the development of resistance remains apossibility and only a few specific insect pests are controllable.Consequently, there remains a long-felt but unfulfilled need to discovernew and effective insect control agents, such as novel δ-endotoxins,that provide an economic benefit to farmers and that are environmentallyacceptable.

SUMMARY OF THE INVENTION

[0005] The present invention addresses the long-standing need for novelinsect control agents. Particularly needed are control agents that aretargeted to economically important insect pests and that efficientlycontrol insect strains resistant to existing insect control agents.Furthermore, agents whose application minimizes the burden on theenvironment are desirable.

[0006] The present invention is drawn to nucleotide sequences isolatedfrom Bacillus thuringiensis ssp. finitimus, and nucleotide sequencessubstantially similar thereto, whose expression result in noveldelta-endotoxins, e.g., Cry26Aa1 and Cry28Aa1, which are toxic toeconomically important pests, particularly plant pests. The invention isfurther drawn to the insecticidal toxins resulting from the expressionof the nucleotide sequences, and to compositions and formulationscontaining the insecticidal toxins, which are capable of inhibiting theability of insect pests to survive, grow or reproduce, or of limitinginsect-related damage or loss in crop plants. The invention is furtherdrawn to a methods of making the toxins and to methods of using thenucleotide sequences, for example in microorganisms to control insectsor in transgenic plants to confer insect resistance, and to methods ofusing the toxins, and compositions and formulations comprising thetoxins, for example applying the toxins, compositions or formulations toinsect infested areas, or to prophylactically treat insect susceptibleareas or plants to confer protection or resistance against harmfulinsects. The toxins can be used in multiple insect control strategies,resulting in maximal efficiency with minimal impact on the environment.

[0007] According to one aspect, the present invention provides anisolated nucleic acid molecule comprising: (a) a nucleotide sequencethat encodes a polypeptide at least 90% identical to SEQ ID NO: 2 or SEQID NO: 4; (b) a nucleotide sequence that encodes SEQ ID NO: 2 or SEQ IDNO: 4; (c) nucleotides 897-4388 of SEQ ID NO: 1 or nucleotides 1129-4458of SEQ ID NO: 3; (d) a consecutive 20 base pair nucleotide portionidentical in sequence to a consecutive 20 base pair portion ofnucleotides 897-4388 of SEQ ID NO: 1 or a consecutive 20 base pairportion of nucleotides 1129-4458 of SEQ ID NO: 3; or (e) a nucleotidesequence whose complement hybridizes under stringent hybridization andwash conditions to nucleotides 897-4388 of SEQ ID NO: 1 or nucleotides1129-4458 of SEQ ID NO: 3; wherein said nucleic acid molecule encodes atoxin that is active against insects.

[0008] The present invention also concerns a chimeric constructcomprising a heterologous promoter sequence operatively linked to anucleic acid molecule of the invention; a recombinant vector comprisingsuch a chimeric construct; and a transgenic host cell comprising such achimeric construct. In one embodiment, the transgenic host cell is abacterial cell; in another embodiment, the transgenic host cell is aplant cell. The present invention further concerns a transgenic plant,such as maize, comprising such a plant cell, as well as seed from such atransgenic plant.

[0009] According to another aspect, the present invention provides atoxin produced by expression of a DNA molecule of the invention. In oneembodiment, the toxin comprises the amino acid sequence set forth as SEQID NO: 2. In another embodiment, the toxin comprises the amino acidsequence set forth as SEQ ID NO: 4. In another embodiment, the toxincomprises an amino acid sequence at least 90% identical to SEQ ID NO: 2.In yet another embodiment, the toxin comprises an amino acid sequence atleast 90% identical to SEQ ID NO: 4. The present invention also concernsa composition comprising an insecticidally effective amount of a toxinaccording to the invention.

[0010] In another aspect, the present invention provides a method ofproducing a toxin that is active against insects, comprising: (a)obtaining a host cell comprising a chimeric construct, which itselfcomprises a heterologous promoter sequence operatively linked to anucleic acid molecule of the invention; and (b) expressing the nucleicacid molecule in the cell, which results in a toxin that is activeagainst insects.

[0011] In a further aspect, the present invention provides a method ofproducing an insect-resistant plant, comprising introducing a nucleicacid molecule of the invention into the plant, wherein the nucleic acidmolecule is expressible in the plant in an effective amount to controlinsects.

[0012] In a still further aspect, the present invention provides amethod of controlling insects comprising delivering to the insects aneffective amount of a toxin according to the present invention.

[0013] Yet another aspect of the present invention is the provision of amethod for mutagenizing a nucleic acid molecule according to the presentinvention, wherein the nucleic acid molecule has been cleaved intopopulation of double-stranded random fragments of a desired size,comprising: (a) adding to the population of double-stranded randomfragments one or more single- or double-stranded oligonucleotides,wherein the oligonucleotides each comprise an area of identity and anarea of heterology to a double-stranded template polynucleotide; (b)denaturing the resultant mixture of double-stranded random fragments andoligonucleotides into single-stranded fragments; (c) incubating theresultant population of single-stranded fragments with a polymeraseunder conditions which result in the annealing of the single-strandedfragments at the areas of identity to form pairs of annealed fragments,the areas of identity being sufficient for one member of a pair to primereplication of the other, thereby forming a mutagenized double-strandedpolynucleotide; and (d) repeating the second and third steps for atleast two further cycles, wherein the resultant mixture in the secondstep of a further cycle includes the mutagenized double-strandedpolynucleotide from the third step of the previous cycle, and whereinthe further cycle forms a further mutagenized double-strandedpolynucleotide.

[0014] Other aspects and advantages of the present invention will becomeapparent to those skilled in the art from a study of the followingdescription of the invention and non-limiting examples.

Definitions

[0015] In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

[0016] “Activity” of the toxins of the invention is meant that thetoxins function as orally active insect control agents, have a toxiceffect, or are able to disrupt or deter insect feeding, which may or maynot cause death of the insect. When a toxin of the invention isdelivered to the insect, the result is typically death of the insect, orthe insect does not feed upon the source that makes the toxin availableto the insect.

[0017] Associated With/Operatively Linked: Refers to two DNA sequencesthat are related physically or functionally. For example, a promoter orregulatory DNA sequence is said to be “associated with” a DNA sequencethat codes for an RNA or a protein if the two sequences are operativelylinked, or situated such that the regulator DNA sequence will affect theexpression level of the coding or structural DNA sequence.

[0018] Chimeric Gene/Chimeric Construct: A recombinant DNA sequence inwhich a promoter or regulatory DNA sequence is operatively linked to, orassociated with, a DNA sequence that codes for an mRNA or which isexpressed as a protein, such that the regulator DNA sequence is able toregulate transcription or expression of the associated DNA sequence. Theregulator DNA sequence of the chimeric gene or chimeric construct is notnormally operatively linked to the associated DNA sequence as found innature.

[0019] Coding Sequence: a nucleic acid sequence that is transcribed intoRNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.Preferably the RNA is then translated in an organism to produce aprotein.

[0020] Complementary: refers to two nucleotide sequences that compriseantiparallel nucleotide sequences capable of pairing with one anotherupon formation of hydrogen bonds between the complementary base residuesin the antiparallel nucleotide sequences.

[0021] To “control” insects means to inhibit, through a toxic effect,the ability of insect pests to survive, grow, feed, and/or reproduce, orto limit insect-related damage or loss in crop plants. To “control”insects may or may not mean killing the insects, although it preferablymeans killing the insects.

[0022] To “deliver” a toxin means that the toxin comes in contact withan insect, resulting in toxic effect and control of the insect. Thetoxin can be delivered in many recognized ways, e.g., orally byingestion by the insect or by contact with the insect via transgenicplant expression, formulated protein composition(s), sprayable proteincomposition(s), a bait matrix, or any other art-recognized toxindelivery system.

[0023] Expression: refers to the transcription and/or translation of anendogenous gene or a transgene in plants. In the case of antisenseconstructs, for example, expression may refer to the transcription ofthe antisense DNA only.

[0024] Expression Cassette: A nucleic acid sequence capable of directingexpression of a particular nucleotide sequence in an appropriate hostcell, comprising a promoter operably linked to the nucleotide sequenceof interest which is operably linked to termination signals. It alsotypically comprises sequences required for proper translation of thenucleotide sequence. The expression cassette comprising the nucleotidesequence of interest may be chimeric, meaning that at least one of itscomponents is heterologous with respect to at least one of its othercomponents. The expression cassette may also be one which is naturallyoccurring but has been obtained in a recombinant form useful forheterologous expression. Typically, however, the expression cassette isheterologous with respect to the host, i.e., the particular nucleic acidsequence of the expression cassette does not occur naturally in the hostcell and must have been introduced into the host cell or an ancestor ofthe host cell by a transformation event. The expression of thenucleotide sequence in the expression cassette may be under the controlof a constitutive promoter or of an inducible promoter which initiatestranscription only when the host cell is exposed to some particularexternal stimulus. In the case of a multicellular organism, such as aplant, the promoter can also be specific to a particular tissue, ororgan, or stage of development.

[0025] Gene: A defined region that is located within a genome and that,besides the aforementioned coding nucleic acid sequence, comprisesother, primarily regulatory, nucleic acid sequences responsible for thecontrol of expression, i.e., transcription and translation of the codingportion. A gene may also comprise other 5′ and 3′ untranslated sequencesand termination sequences. Further elements that may be present are, forexample, introns.

[0026] Heterologous DNA Sequence: The terms “heterologous DNA sequence”,“exogenous DNA segment” or “heterologous nucleic acid,” as used herein,each refer to a sequence that originates from a source foreign to theparticular host cell or, if from the same source, is modified from itsoriginal form. Thus, a heterologous gene in a host cell includes a genethat is endogenous to the particular host cell but has been modifiedthrough, for example, the use of DNA shuffling. The terms also includesnon-naturally occurring multiple copies of a naturally occurring DNAsequence. Thus, the terms refer to a DNA segment that is foreign orheterologous to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is not ordinarilyfound. Exogenous DNA segments are expressed to yield exogenouspolypeptides.

[0027] Homologous DNA Sequence: A DNA sequence naturally associated witha host cell into which it is introduced.

[0028] The terms “identical” or percent “identity” in the context of twoor more nucleic acid or protein sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described below or by visualinspection.

[0029] “Insecticidal” is defined as a toxic biological activity capableof controlling insects, preferably by killing them.

[0030] Isocoding: A nucleic acid sequence is isocoding with a referencenucleic acid sequence when the nucleic acid sequence encodes apolypeptide having the same amino acid sequence as the polypeptideencoded by the reference nucleic acid sequence.

[0031] Isolated: In the context of the present invention, an isolatednucleic acid molecule or an isolated enzyme is a nucleic acid moleculeor enzyme that, by the hand of man, exists apart from its nativeenvironment and is therefore not a product of nature. An isolatednucleic acid molecule or enzyme may exist in a purified form or mayexist in a non-native environment such as, for example, a recombinanthost cell.

[0032] Minimal Promoter: a promoter element, particularly a TATAelement, that is inactive or has greatly reduced promoter activity inthe absence of upstream activation. In the presence of a suitabletranscription factor, a minimal promoter functions to permittranscription.

[0033] Native: refers to a gene that is present in the genome of anuntransformed cell.

[0034] Naturally occurring: the term “naturally occurring” is used todescribe an object that can be found in nature as distinct from beingartificially produced by man. For example, a protein or nucleotidesequence present in an organism (including a virus), which can beisolated from a source in nature and which has not been intentionallymodified by man in the laboratory, is naturally occurring.

[0035] Nucleic acid: the term “nucleic acid” refers todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides which have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g. degenerate codon substitutions) and complementarysequences and as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka etal., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell.Probes 8: 91-98 (1994)). The terms “nucleic acid” or “nucleic acidsequence” may also be used interchangeably with gene, cDNA, and mRNAencoded by a gene. In the context of the present invention, the nucleicacid molecule is preferably a segment of DNA. Nucleotides are indicatedby their bases by the following standard abbreviations: adenine (A),cytosine (C), thymine (T), and guanine (G).

[0036] ORF: Open Reading Frame.

[0037] Plant: Any whole plant.

[0038] Plant Cell: Structural and physiological unit of a plant,comprising a protoplast and a cell wall. The plant cell may be in formof an isolated single cell or a cultured cell, or as a part of higherorganized unit such as, for example, a plant tissue, a plant organ, or awhole plant.

[0039] Plant Cell Culture: Cultures of plant units such as, for example,protoplasts, cell culture cells, cells in plant tissues, pollen, pollentubes, ovules, embryo sacs, zygotes and embryos at various stages ofdevelopment.

[0040] Plant Material: Refers to leaves, stems, roots, flowers or flowerparts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell ortissue cultures, or any other part or product of a plant.

[0041] Plant Organ: A distinct and visibly structured and differentiatedpart of a plant such as a root, stem, leaf, flower bud, or embryo.

[0042] Plant tissue: A group of plant cells organized into a structuraland functional unit. Any tissue of a plant in planta or in culture isincluded. This term includes, but is not limited to, whole plants, plantorgans, plant seeds, tissue culture and any groups of plant cellsorganized into structural and/or functional units. The use of this termin conjunction with, or in the absence of, any specific type of planttissue as listed above or otherwise embraced by this definition is notintended to be exclusive of any other type of plant tissue.

[0043] Promoter: An untranslated DNA sequence upstream of the codingregion that contains the binding site for RNA polymerase II andinitiates transcription of the DNA. The promoter region may also includeother elements that act as regulators of gene expression.

[0044] Protoplast: An isolated plant cell without a cell wall or withonly parts of the cell wall.

[0045] Purified: the term “purified,” when applied to a nucleic acid orprotein, denotes that the nucleic acid or protein is essentially free ofother cellular components with which it is associated in the naturalstate. It is preferably in a homogeneous state although it can be ineither a dry or aqueous solution. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. The term “purified” denotes that a nucleic acidor protein gives rise to essentially one band in an electrophoretic gel.Particularly, it means that the nucleic acid or protein is at leastabout 50% pure, more preferably at least about 85% pure, and mostpreferably at least about 99% pure.

[0046] Recombinant DNA molecule: a combination of DNA molecules that arejoined together using recombinant DNA technology.

[0047] Regulatory Elements: Sequences involved in controlling theexpression of a nucleotide sequence. Regulatory elements comprise apromoter operably linked to the nucleotide sequence of interest andtermination signals. They also typically encompass sequences requiredfor proper translation of the nucleotide sequence.

[0048] Selectable marker gene: a gene whose expression in a plant cellgives the cell a selective advantage. The selective advantage possessedby the cells transformed with the selectable marker gene may be due totheir ability to grow in the presence of a negative selective agent,such as an antibiotic or a herbicide, compared to the growth ofnon-transformed cells. The selective advantage possessed by thetransformed cells, compared to non-transformed cells, may also be due totheir enhanced or novel capacity to utilize an added compound as anutrient, growth factor or energy source. Selectable marker gene alsorefers to a gene or a combination of genes whose expression in a plantcell gives the cell both, a negative and a positive selective advantage.

[0049] Substantially identical: the phrase “substantially identical,” inthe context of two nucleic acid or protein sequences, refers to two ormore sequences or subsequences that have at least 60%, preferably 80%,more preferably 90-95%, and most preferably at least 99% nucleotide oramino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. Preferably, thesubstantial identity exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably the sequences aresubstantially identical over at least about 150 residues. In a mostpreferred embodiment, the sequences are substantially identical over theentire length of the coding regions. Furthermore, substantiallyidentical nucleic acid or protein sequences perform substantially thesame function.

[0050] For sequence comparison, typically one sequence acts as areference sequence to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer, subsequence coordinates are designated if necessary,and sequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

[0051] Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl.Math. 2: 482 (1981), by the homology alignment algorithm of Needleman &Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similaritymethod of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988),by computerized implementations of these algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Dr., Madison, Wis.), or by visual inspection(see generally, Ausubel et al., infra).

[0052] One example of an algorithm that is suitable for determiningpercent sequence identity and sequence similarity is the BLASTalgorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST-analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., 1990). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.USA 89: 10915 (1989)).

[0053] In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90: 5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleic acidsequence to the reference nucleic acid sequence is less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

[0054] Another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

[0055] “Stringent hybridization conditions” and “stringent washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays” Elsevier,New York. Generally, highly stringent hybridization and wash conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.Typically, under “stringent conditions” a probe will hybridize to itstarget subsequence, but to no other sequences.

[0056] The T_(m) is the temperature (under defined ionic strength andpH) at which 50% of the target sequence hybridizes to a perfectlymatched probe. Very stringent conditions are selected to be equal to theT_(m) for a particular probe. An example of stringent hybridizationconditions for hybridization of complementary nucleic acids which havemore than 100 complementary residues on a filter in a Southern ornorthern blot is 50% formamide with 1 mg of heparin at 42° C., with thehybridization being carried out overnight. An example of highlystringent wash conditions is 0.1 5M NaCl at 72° C. for about 15 minutes.An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for15 minutes (see, Sambrook, infra, for a description of SSC buffer).Often, a high stringency wash is preceded by a low stringency wash toremove background probe signal. An example medium stringency wash for aduplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15minutes. An example low stringency wash for a duplex of, e.g., more than100 nucleotides, is 4-6×SSC at 40° C. for 15 minutes. For short probes(e.g., about 10 to 50 nucleotides), stringent conditions typicallyinvolve salt concentrations of less than about 1.0M Na ion, typicallyabout 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to8.3, and the temperature is typically at least about 30° C. Stringentconditions can also be achieved with the addition of destabilizingagents such as formamide. In general, a signal to noise ratio of 2× (orhigher) than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the proteins that theyencode are substantially identical. This occurs, e.g., when a copy of anucleic acid is created using the maximum codon degeneracy permitted bythe genetic code.

[0057] The following are examples of sets of hybridization/washconditions that may be used to clone homologous nucleotide sequencesthat are substantially identical to reference nucleotide sequences ofthe present invention: a reference nucleotide sequence preferablyhybridizes to the reference nucleotide sequence in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC,0.1% SDS at 50° C., more desirably in 7% sodium dodecyl sulfate (SDS),0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50°C., more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 0.5×SSC, 0.1% SDS at 50° C.,preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. with washing in 0.1×SSC, 0.1% SDS at 50° C., more preferably in7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. withwashing in 0.1×SSC, 0.1% SDS at 65° C.

[0058] A further indication that two nucleic acid sequences or proteinsare substantially identical is that the protein encoded by the firstnucleic acid is immunologically cross reactive with, or specificallybinds to, the protein encoded by the second nucleic acid. Thus, aprotein is typically substantially identical to a second protein, forexample, where the two proteins differ only by conservativesubstitutions.

[0059] The phrase “specifically (or selectively) binds to an antibody,”or “specifically (or selectively) immunoreactive with,” when referringto a protein or peptide, refers to a binding reaction which isdeterminative of the presence of the protein in the presence of aheterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antibodies bind to aparticular protein and do not bind in a significant amount to otherproteins present in the sample. Specific binding to an antibody undersuch conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, antibodies raised tothe protein with the amino acid sequence encoded by any of the nucleicacid sequences of the invention can be selected to obtain antibodiesspecifically immunoreactive with that protein and not with otherproteins except for polymorphic variants. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassays,Western blots, or immunohistochemistry are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Publications, New York “Harlow and Lane”), for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity. Typically a specific or selective reactionwill be at least twice background signal or noise and more typicallymore than 10 to 100 times background.

[0060] “Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acid sequences that encode identical oressentially identical amino acid sequences, or where the nucleic acidsequence does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenpolypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGGall encode the amino acid arginine. Thus, at every position where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded protein.Such nucleic acid variations are “silent variations” which are onespecies of “conservatively modified variations.” Every nucleic acidsequence described herein which encodes a protein also describes everypossible silent variation, except where otherwise noted. One of skillwill recognize that each codon in a nucleic acid (except ATG, which isordinarily the only codon for methionine) can be modified to yield afunctionally identical molecule by standard techniques. Accordingly,each “silent variation” of a nucleic acid which encodes a protein isimplicit in each described sequence.

[0061] Furthermore, one of skill will recognize that individualsubstitutions deletions or additions that alter, add or delete a singleamino acid or a small percentage of amino acids (typically less than 5%,more typically less than 1%) in an encoded sequence are “conservativelymodified variations,” where the alterations result in the substitutionof an amino acid with a chemically similar amino acid. Conservativesubstitution tables providing functionally similar amino acids are wellknown in the art. The following five groups each contain amino acidsthat are conservative substitutions for one another: Aliphatic: Glycine(G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic:Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing:Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine(H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N),Glutamine (Q). See also, Creighton (1984) Proteins, W. H. Freeman andCompany. In addition, individual substitutions, deletions or additionswhich alter, add or delete a single amino acid or a small percentage ofamino acids in an encoded sequence are also “conservatively modifiedvariations.”

[0062] A “subsequence” refers to a sequence of nucleic acids or aminoacids that comprise a part of a longer sequence of nucleic acids oramino acids (e.g., protein) respectively.

[0063] Nucleic acids are “elongated” when additional nucleotides (orother analogous molecules) are incorporated into the nucleic acid. Mostcommonly, this is performed with a polymerase (e.g., a DNA polymerase),e.g., a polymerase which adds sequences at the 3′ terminus of thenucleic acid.

[0064] Two nucleic acids are “recombined” when sequences from each ofthe two nucleic acids are combined in a progeny nucleic acid. Twosequences are “directly” recombined when both of the nucleic acids aresubstrates for recombination. Two sequences are “indirectly recombined”when the sequences are recombined using an intermediate such as across-over oligonucleotide. For indirect recombination, no more than oneof the sequences is an actual substrate for recombination, and in somecases, neither sequence is a substrate for recombination.

[0065] A “specific binding affinity” between two molecules, for example,a ligand and a receptor, means a preferential binding of one moleculefor another in a mixture of molecules. The binding of the molecules canbe considered specific if the binding affinity is about 1×10⁴ M⁻¹ toabout 1×10⁶ M⁻¹ or greater.

[0066] “Synthetic” refers to a nucleotide sequence comprising structuralcharacters that are not present in the natural sequence. For example, anartificial sequence that resembles more closely the G+C content and thenormal codon distribution of dicot and/or monocot genes is said to besynthetic.

[0067] Transformation: a process for introducing heterologous DNA into ahost cell or organism.

[0068] “Transformed,” “transgenic,” and “recombinant” refer to a hostorganism such as a bacterium or a plant into which a heterologousnucleic acid molecule has been introduced. The nucleic acid molecule canbe stably integrated into the genome of the host or the nucleic acidmolecule can also be present as an extrachromosomal molecule. Such anextrachromosomal molecule can be auto-replicating. Transformed cells,tissues, or plants are understood to encompass not only the end productof a transformation process, but also transgenic progeny thereof. A“non-transformed,” “non-transgenic,” or “non-recombinant” host refers toa wild-type organism, e.g., a bacterium or plant, which does not containthe heterologous nucleic acid molecule.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

[0069] SEQ ID NO: 1 is the DNA sequence of clone pF1 isolated fromBacillus thuringiensis ssp. finitimus that comprises the coding sequenceof the cry26Aa1 gene.

[0070] SEQ ID NO: 2 is the amino acid sequence of the Cry26Aa1delta-endotoxin.

[0071] SEQ ID NO: 3 is the DNA sequence of clone pF2 isolated fromBacillus thuringiensis ssp. finitimus that comprises the coding sequenceof the cry28Aa1 gene.

[0072] SEQ ID NO: 4 is the amino acid sequence of the Cry28Aa1delta-endotoxin.

[0073] SEQ ID NO: 5 is a putative vegetative promoter sequence.

DETAILED DESCRIPTION OF THE INVENTION Novel Nucleic Acid Sequences whoseExpression Results in Insecticidal Toxins

[0074] This invention relates to nucleic acid sequences whose expressionresults in novel delta-endotoxins, and to the making and using of thetoxins to control insect pests. The nucleic acid sequences are isolatedfrom Bacillus thuringiensis ssp. finitimus, which forms insecticidalcrystal bodies either outside or inside of exosporium. In particular,the present invention provides genes cry26Aa1 and cry28Aa1 cloned fromBacillus thuringiensis ssp. finitimus strain B-1166 VKPM. The deducedamino acid sequence of the cry26Aa1 gene product includes 7 residuesdetermined as an N-terminal part of a chymotrypsin treateddelta-endotoxin isolated from the same strain. Neither BtI nor BtIIpromoter sequences are found upstream of the open reading frames in bothgenes. Southern hybridization shows that the surroundings of both genesat least 3 kb upstream and downstream of the open reading frames areunique. See also, Wojciechowska et al., FEBS Lett. 453(1-2): 46-48(1999), incorporated herein by reference.

[0075] In a preferred embodiment, the invention encompasses an isolatednucleic acid molecule comprising: (a) a nucleotide sequencesubstantially identical to nucleotides 897-4388 of SEQ ID NO: 1 ornucleotides 1129-4458 of SEQ ID NO: 3; or (b) a nucleotide sequenceisocoding with the nucleotide sequence of (a); or (c) a 20 base pairnucleotide portion identical in sequence to a consecutive 20 base pairportion of nucleotides 897-4388 of SEQ ID NO: 1 or a consecutive 20 basepair portion of nucleotides 1129-4458 of SEQ ID NO: 3; whereinexpression of said nucleic acid molecule results in a toxin that isactive against insects. The present invention also encompassesrecombinant vectors comprising the nucleic acid sequences of thisinvention. In such vectors, the nucleic acid sequences are preferablycomprised in expression cassettes comprising regulatory elements forexpression of the nucleotide sequences in a host cell capable ofexpressing the nucleotide sequences. Such regulatory elements usuallycomprise promoter and termination signals and preferably also compriseelements allowing efficient translation of polypeptides encoded by thenucleic acid sequences of the present invention. Vectors comprising thenucleic acid sequences are usually capable of replication in particularhost cells, preferably as extrachromosomal molecules, and are thereforeused to amplify the nucleic acid sequences of this invention in the hostcells. In one embodiment, host cells for such vectors aremicroorganisms, such as bacteria, in particular E. coli. In anotherembodiment, host cells for such recombinant vectors are endophytes orepiphytes. A preferred host cell for such vectors is a eukaryotic cell,such as a yeast, a plant cell, or an insect cell. Plant cells such asmaize cells are most preferred host cells. In another preferredembodiment, such vectors are viral vectors and are used for replicationof the nucleotide sequences in particular host cells, e.g. insect cellsor plant cells. Recombinant vectors are also used for transformation ofthe nucleotide sequences of this invention into host cells, whereby thenucleotide sequences are stably integrated into the DNA of such hostcells. In one, such host cells are prokaryotic cells. In a preferredembodiment, such host cells are eukaryotic cells, such as yeast cells,insect cells, or plant cells. In a most preferred embodiment, the hostcells are plant cells, such as maize cells.

[0076] The nucleotide sequences of the invention can be isolated usingthe techniques described in the examples below, or by PCR using thesequences set forth in the sequence listing as the basis forconstructing PCR primers. For example, oligonucleotides having thesequence of approximately the first and last 20-25 consecutivenucleotides of the cry26Aa1 coding sequence set forth in SEQ ID NO: 1(e.g., nucleotides 897-916 and 4369-4388 of SEQ ID NO: 1) can be used asPCR primers to amplify the cry26Aa1 coding sequence (nucleotides897-4388 of SEQ ID NO: 1) directly from the source strain (Bacillusthuringiensis ssp. finitimus). The cry28Aa1 gene sequence can also beamplified directly from the source strain in an analogous manner.Furthermore, homologues of the cry26Aa1 and cry28Aa1 can be isolatedfrom other Bt strains using these sequences as primers.

[0077] In further embodiments, the nucleotide sequences of the inventioncan be modified by incorporation of random mutations in a techniqueknown as in-vitro recombination or DNA shuffling. This technique isdescribed in Stemmer et al., Nature 370: 389-391 (1994) and U.S. Pat.No. 5,605,793, which are incorporated herein by reference. Millions ofmutant copies of a nucleotide sequence are produced based on an originalnucleotide sequence of this invention and variants with improvedproperties, such as increased insecticidal activity, enhanced stability,or different specificity or range of target insect pests are recovered.The method encompasses forming a mutagenized double-strandedpolynucleotide from a template double-stranded polynucleotide comprisinga nucleotide sequence of this invention, wherein the templatedouble-stranded polynucleotide has been cleaved intodouble-stranded-random fragments of a desired size, and comprises thesteps of adding to the resultant population of double-stranded randomfragments one or more single or double-stranded oligonucleotides,wherein said oligonucleotides comprise an area of identity and an areaof heterology to the double-stranded template polynucleotide; denaturingthe resultant mixture of double-stranded random fragments andoligonucleotides into single-stranded fragments; incubating theresultant population of single-stranded fragments with a polymeraseunder conditions which result in the annealing of said single-strandedfragments at said areas of identity to form pairs of annealed fragments,said areas of identity being sufficient for one member of a pair toprime replication of the other, thereby forming a mutagenizeddouble-stranded polynucleotide; and repeating the second and third stepsfor at least two further cycles, wherein the resultant mixture in thesecond step of a further cycle includes the mutagenized double-strandedpolynucleotide from the third step of the previous cycle, and thefurther cycle forms a further mutagenized double-strandedpolynucleotide. In a preferred embodiment, the concentration of a singlespecies of double-stranded random fragment in the population ofdouble-stranded random fragments is less than 1% by weight of the totalDNA. In a further preferred embodiment, the template double-strandedpolynucleotide comprises at least about 100 species of polynucleotides.In another preferred embodiment, the size of the double-stranded randomfragments is from about 5 bp to 5 kb. In a further preferred embodiment,the fourth step of the method comprises repeating the second and thethird steps for at least 10 cycles.

Expression of the Nucleotide Sequences in Heterologous Microbial Hosts

[0078] As biological insect control agents, the insecticidal toxins areproduced by expression of the nucleotide sequences in heterologous hostcells capable of expressing the nucleotide sequences. In a firstembodiment, one of the nucleotide sequences of the invention is insertedinto an appropriate expression cassette, comprising a promoter andtermination signals. Expression of the nucleotide sequence isconstitutive, or an inducible promoter responding to various types ofstimuli to initiate transcription is used. In a preferred embodiment,the cell in which the toxin is expressed is a microorganism, such as avirus, a bacteria, or a fungus. In a preferred embodiment, a virus, suchas a baculovirus, contains a nucleotide sequence of the invention in itsgenome and expresses large amounts of the corresponding insecticidaltoxin after infection of appropriate eukaryotic cells that are suitablefor virus replication and expression of the nucleotide sequence. Theinsecticidal toxin thus produced is used as an insecticidal agent.Alternatively, baculoviruses engineered to include the nucleotidesequence are used to infect insects in-vivo and kill them either byexpression of the insecticidal toxin or by a combination of viralinfection and expression of the insecticidal toxin.

[0079] Bacterial cells are also hosts for the expression of thenucleotide sequences of the invention. In a preferred embodiment,non-pathogenic symbiotic bacteria, which are able to live and replicatewithin plant tissues, so-called endophytes, or non-pathogenic symbioticbacteria, which are capable of colonizing the phyllosphere or therhizosphere, so-called epiphytes, are used. Such bacteria includebacteria of the genera Agrobacterium, Alcaligenes, Azospirillum,Azotobacter, Bacillus, Clavibacter, Enterobacter, Erwinia, Flavobacter,Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomyces andXanthomonas. Symbiotic fungi, such as Trichoderma and Gliocladium arealso possible hosts for expression of the inventive nucleotide sequencesfor the same purpose.

[0080] Techniques for these genetic manipulations are specific for thedifferent available hosts and are known in the art. For example, theexpression vectors pKK223-3 and pKK223-2 can be used to expressheterologous genes in E. coli, either in transcriptional ortranslational fusion, behind the tac or trc promoter. For the expressionof operons encoding multiple ORFs, the simplest procedure is to insertthe operon into a vector such as pKK223-3 in transcriptional fusion,allowing the cognate ribosome binding site of the heterologous genes tobe used. Techniques for overexpression in gram-positive species such asBacillus are also known in the art and can be used in the context ofthis invention (Quax et al. In.: Industrial Microorganisms: Basic andApplied Molecular Genetics, Eds. Baltz et al., American Society forMicrobiology, Washington (1993)). Alternate systems for overexpressionrely for example, on yeast vectors and include the use of Pichia,Saccharomyces and Kluyveromyces (Sreekrishna, In: Industrialmicroorganisms: basic and applied molecular genetics, Baltz, Hegeman,and Skatrud eds., American Society for Microbiology, Washington (1993);Dequin & Barre, Biotechnology 12:173-177 (1994); van den Berg et al.,Biotechnology 8:135-139 (1990)).

[0081] In another preferred embodiment, at least one of the describednucleotide sequences is transferred to and expressed in Pseudomonasfluorescens strain CGA267356 (described in the published application EU0 472 494 and in WO 94/01561) which has biocontrol characteristics. Inanother preferred embodiment, a nucleotide sequence of the invention istransferred to Pseudomonas aureofaciens strain 30-84 which also hasbiocontrol characteristics. Expression in heterologous biocontrolstrains requires the selection of vectors appropriate for replication inthe chosen host and a suitable choice of promoter. Techniques are wellknown in the art for expression in gram-negative and gram-positivebacteria and fungi.

Expression of the Nucleotide Sequences in Plant Tissue

[0082] In a particularly preferred embodiment, at least one of theinsecticidal toxins of the invention is expressed in a higher organism,e.g., a plant. In this case, transgenic plants expressing effectiveamounts of the toxins protect themselves from insect pests. When theinsect starts feeding on such a transgenic plant, it also ingests theexpressed toxins. This will deter the insect from further biting intothe plant tissue or may even harm or kill the insect. A nucleotidesequence of the present invention is inserted into an expressioncassette, which is then preferably stably integrated in the genome ofsaid plant. In another preferred embodiment, the nucleotide sequence isincluded in a non-pathogenic self-replicating virus. Plants transformedin accordance with the present invention may be monocots or dicots andinclude, but are not limited to, maize, wheat, barley, rye, sweetpotato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli,turnip, radish, spinach, asparagus, onion, garlic, pepper, celery,squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, plum,cherry, peach, nectarine, apricot, strawberry, grape, raspberry,blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato,sorghum, sugarcane, sugarbeet, sunflower, grapeseed, clover, tobacco,carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis,and woody plants such as coniferous and deciduous trees.

[0083] Once a desired nucleotide sequence has been transformed into aparticular plant species, it may be propagated in that species or movedinto other varieties of the same species, particularly includingcommercial varieties, using traditional breeding techniques.

[0084] A nucleotide sequence of this invention is preferably expressedin transgenic plants, thus causing the biosynthesis of the correspondingtoxin in the transgenic plants. In this way, transgenic plants withenhanced resistance to insects are generated. For their expression intransgenic plants, the nucleotide sequences of the invention may requiremodification and optimization. Although in many cases genes frommicrobial organisms can be expressed in plants at high levels withoutmodification, low expression in transgenic plants may result frommicrobial nucleotide sequences having codons that are not preferred inplants. It is known in the art that all organisms have specificpreferences for codon usage, and the codons of the nucleotide sequencesdescribed in this invention can be changed to conform with plantpreferences, while maintaining the amino acids encoded thereby.Furthermore, high expression in plants is best achieved from codingsequences that have at least 35% about GC content, preferably more thanabout 45%, more preferably more than about 50%, and most preferably morethan about 60%. Microbial nucleotide sequences which have low GCcontents may express poorly in plants due to the existence of ATTTAmotifs which may destabilize messages, and AATAAA motifs which may causeinappropriate polyadenylation. Although preferred gene sequences may beadequately expressed in both monocotyledonous and dicotyledonous plantspecies, sequences can be modified to account for the specific codonpreferences and GC content preferences of monocotyledons or dicotyledonsas these preferences have been shown to differ (Murray et al. Nucl.Acids Res. 17: 477-498 (1989)). In addition, the nucleotide sequencesare screened for the existence of illegitimate splice sites that maycause message truncation. All changes required to be made within thenucleotide sequences such as those described above are made using wellknown techniques of site directed mutagenesis, PCR, and synthetic geneconstruction using the methods described in the published patentapplications EP 0 385 962 (to Monsanto), EP 0 359 472 (to Lubrizol, andWO 93/07278 (to Ciba-Geigy).

[0085] For efficient initiation of translation, sequences adjacent tothe initiating methionine may require modification. For example, theycan be modified by the inclusion of sequences known to be effective inplants. Joshi has suggested an appropriate consensus for plants (NAR 15:6643-6653 (1987)) and Clontech suggests a further consensus translationinitiator (1993/1994 catalog, page 210). These consensuses are suitablefor use with the nucleotide sequences of this invention. The sequencesare incorporated into constructions comprising the nucleotide sequences,up to and including the ATG (whilst leaving the second amino acidunmodified), or alternatively up to and including the GTC subsequent tothe ATG (with the possibility of modifying the second amino acid of thetransgene).

[0086] Expression of the nucleotide sequences in transgenic plants isdriven by promoters shown to be functional in plants. The choice ofpromoter will vary depending on the temporal and spatial requirementsfor expression, and also depending on the target species. Thus,expression of the nucleotide sequences of this invention in leaves, inears, in inflorescences (e.g. spikes, panicles, cobs, etc.), in roots,and/or seedlings is preferred. In many cases, however, protectionagainst more than one type of insect pest is sought, and thus expressionin multiple tissues is desirable. Although many promoters fromdicotyledons have been shown to be operational in monocotyledons-andvice versa, ideally dicotyledonous promoters are selected for expressionin dicotyledons, and monocotyledonous promoters for expression inmonocotyledons. However, there is no restriction to the provenance ofselected promoters; it is sufficient that they are operational indriving the expression of the nucleotide sequences in the desired cell.

[0087] Preferred promoters that are expressed constitutively includepromoters from genes encoding actin or ubiquitin and the CaMV 35S and19S promoters. The nucleotide sequences of this invention can also beexpressed under the regulation of promoters that are chemicallyregulated. This enables the insecticidal toxins to be synthesized onlywhen the crop plants are treated with the inducing chemicals. Preferredtechnology for chemical induction of gene expression is detailed in thepublished application EP 0 332 104 (to Ciba-Geigy) and U.S. Pat. No.5,614,395. A preferred promoter for chemical induction is the tobaccoPR-1a promoter.

[0088] A preferred category of promoters is that which is woundinducible. Numerous promoters have been described which are expressed atwound sites and also at the sites of phytopathogen infection. Ideally,such a promoter should only be active locally at the sites of infection,and in this way the insecticidal toxins only accumulate in cells whichneed to synthesize the insecticidal toxins to kill the invading insectpest. Preferred promoters of this kind include those described byStanford et al. Mol. Gen. Genet. 215: 200-208 (1989), Xu et al. PlantMolec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1: 151-158(1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Fireket al. Plant Molec. Biol. 22: 129-142 (1993), and Warner et al. Plant J.3: 191-201 (1993).

[0089] Preferred tissue specific expression patterns include greentissue specific, root specific, stem specific, and flower specific.Promoters suitable for expression in green tissue include many whichregulate genes involved in photosynthesis and many of these have beencloned from both monocotyledons and dicotyledons. A preferred promoteris the maize PEPC promoter from the phosphoenol carboxylase gene(Hudspeth & Grula, Plant Molec. Biol. 12: 579-589 (1989)). A preferredpromoter for root specific expression is that described by de Framond(FEBS 290: 103-106 (1991); EP 0 452 269 to Ciba-Geigy). A preferred stemspecific promoter is that described in U.S. Pat. No. 5,625,136 (toCiba-Geigy) and which drives expression of the maize trpA gene.

[0090] Especially preferred embodiments of the invention are transgenicplants expressing at least one of the nucleotide sequences of theinvention in a root-preferred or root-specific fashion. Furtherpreferred embodiments are transgenic plants expressing the nucleotidesequences in a wound-inducible or pathogen infection-inducible manner.

[0091] In addition to the selection of a suitable promoter,constructions for expression of an insecticidal toxin in plants requirean appropriate transcription terminator to be attached downstream of theheterologous nucleotide sequence. Several such terminators are availableand known in the art (e.g. tm1 from CaMV, E9 from rbcS). Any availableterminator known to function in plants can be used in the context ofthis invention.

[0092] Numerous other sequences can be incorporated into expressioncassettes described in this invention. These include sequences whichhave been shown to enhance expression such as intron sequences (e.g.from Adh1 and bronze1) and viral leader sequences (e.g. from TMV, MCMVand AMV).

[0093] It may be preferable to target expression of the nucleotidesequences of the present invention to different cellular localizationsin the plant. In some cases, localization in the cytosol may bedesirable, whereas in other cases, localization in some subcellularorganelle may be preferred. Subcellular localization of transgeneencoded enzymes is undertaken using techniques well known in the art.Typically, the DNA encoding the target peptide from a knownorganelle-targeted gene product is manipulated and fused upstream of thenucleotide sequence. Many such target sequences are known for thechloroplast and their functioning in heterologous constructions has beenshown. The expression of the nucleotide sequences of the presentinvention is also targeted to the endoplasmic reticulum or to thevacuoles of the host cells. Techniques to achieve this are well-known inthe art.

[0094] Vectors suitable for plant transformation are described elsewherein this specification. For Agrobacterium-mediated transformation, binaryvectors or vectors carrying at least one T-DNA border sequence aresuitable, whereas for direct gene transfer any vector is suitable andlinear DNA containing only the construction of interest may bepreferred. In the case of direct gene transfer, transformation with asingle DNA species or co-transformation can be used (Schocher et al.Biotechnology 4: 1093-1096 (1986)). For both direct gene transfer andAgrobacterium-mediated transfer, transformation is usually (but notnecessarily) undertaken with a selectable marker which may provideresistance to an antibiotic (kanamycin, hygromycin or methotrexate) or aherbicide (basta). Examples of such markers are neomycinphosphotransferase, hygromycin phosphotransferase, dihydrofolatereductase, phosphinothricin acetyltransferase, 2,2-dichloroproprionicacid dehalogenase, acetohydroxyacid synthase,5-enolpyruvyl-shikimate-phosphate synthase, haloarylnitrilase,protoporhyrinogen oxidase, acetyl-coenzyme A carboxylase,dihydropteroate synthase, chloramphenicol acetyl transferase, andβ-glucuronidase. The choice of selectable or screenable marker for planttransformation is not, however, critical to the invention.

[0095] The recombinant DNA described above can be introduced into theplant cell in a number of art-recognized ways. Those skilled in the artwill appreciate that the choice of method might depend on the type ofplant targeted for transformation. Suitable methods of transformingplant cells include microinjection (Crossway et al., BioTechniques4:320-334 (1986)), electroporation (Riggs et al., Proc. Natl. Acad. Sci.USA 83:5602-5606 (1986), Agrobacterium-mediated transformation (Hincheeet al., Biotechnology 6:915-921 (1988); See also, Ishida et al., NatureBiotechnology 14:745-750 (June 1996) for maize transformation), directgene transfer (Paszkowski et al., EMBO J. 3:2717-2722 (1984);Hayashimoto et al., Plant Physiol. 93:857-863 (1990)(rice)), andballistic particle acceleration using devices available from Agracetus,Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del. (see, forexample, Sanford et al., U.S. Pat. No. 4,945,050; and McCabe et al.,Biotechnology 6:923-926 (1988)). See also, Weissinger et al., AnnualRev. Genet. 22:421-477 (1988); Sanford et al., Particulate Science andTechnology 5:27-37 91987)(onion); Svab et al., Proc. Natl. Acad. Sci.USA 87: 8526-8530 (1990) (tobacco chloroplast); Christou et al., PlantPhysiol. 87:671-674 (1988)(soybean); McCabe et al., Bio/Technology6:923-926 (1988)(soybean); Klein et al., Proc. Natl. Acad. Sci. USA,85:4305-4309 (1988)(maize); Klein et al., Bio/Technology 6:559-563(1988) (maize); Klein et al., Plant Physiol. 91:440-444 (1988) (maize);Fromm et al., Bio/Technology 8:833-839 (1990); and Gordon-Kamm et al.,Plant Cell 2: 603-618 (1990) (maize); Koziel et al., Biotechnology 11:194-200 (1993) (maize); Shimamoto et al., Nature 338: 274-277 (1989)(rice); Christou et al., Biotechnology 9: 957-962 (1991) (rice); Dattaet al., Bio/Technology 8:736-740 (1990) (rice); European PatentApplication EP 0 332 581 (orchardgrass and other Pooideae); Vasil etal., Biotechnology 11: 1553-1558 (1993) (wheat); Weeks et al., PlantPhysiol. 102: 1077-1084 (1993) (wheat); Wan et al., Plant Physiol. 104:37-48 (1994) (barley); Jahne et al., Theor. Appl. Genet. 89:525-533(1994)(barley); Umbeck et al., Bio/Technology 5: 263-266 (1987)(cotton); Casas et al., Proc. Natl. Acad. Sci. USA 90:11212-11216(December 1993) (sorghum); Somers et al., Bio/Technology 10:1589-1594(December 1992) (oat); Torbert et al., Plant Cell Reports 14:635-640(1995) (oat); Weeks et al., Plant Physiol. 102:1077-1084 (1993) (wheat);Chang et al., WO 94/13822 (wheat) and Nehra et al., The Plant Journal5:285-297 (1994) (wheat). A particularly preferred set of embodimentsfor the introduction of recombinant DNA molecules into maize bymicroprojectile bombardment can be found in Koziel et al., Biotechnology11: 194-200 (1993), Hill et al., Euphytica 85:119-123 (1995) and Kozielet al., Annals of the New York Academy of Sciences 792:164-171 (1996).An additional preferred embodiment is the protoplast transformationmethod for maize as disclosed in EP 0 292 435. Transformation of plantscan be undertaken with a single DNA species or multiple DNA species(i.e. co-transformation) and both these techniques are suitable for usewith the peroxidase coding sequence.

[0096] In another preferred embodiment, a nucleotide sequence of thepresent invention is directly transformed into the plastid genome. Amajor advantage of plastid transformation is that plastids are generallycapable of expressing bacterial genes without substantial modification,and plastids are capable of expressing multiple open reading framesunder control of a single promoter. Plastid transformation technology isextensively described in U.S. Pat. Nos. 5,451,513, 5,545,817, and5,545,818, in PCT application no. WO 95/16783, and in McBride et al.(1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305. The basic technique forchloroplast transformation involves introducing regions of clonedplastid DNA flanking a selectable marker together with the gene ofinterest into a suitable target tissue, e.g., using biolistics orprotoplast transformation (e.g., calcium chloride or PEG mediatedtransformation). The 1 to 1.5 kb flanking regions, termed targetingsequences, facilitate homologous recombination with the plastid genomeand thus allow the replacement or modification of specific regions ofthe plastome. Initially, point mutations in the chloroplast 16S rRNA andrps12 genes conferring resistance to spectinomycin and/or streptomycinare utilized as selectable markers for transformation (Svab, Z.,Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87,8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45).This resulted in stable homoplasmic transformants at a frequency ofapproximately one per 100 bombardments of target leaves. The presence ofcloning sites between these markers allowed creation of a plastidtargeting vector for introduction of foreign genes (Staub, J. M., andMaliga, P. (1993) EMBO J. 12, 601-606). Substantial increases intransformation frequency are obtained by replacement of the recessiverRNA or r-protein antibiotic resistance genes with a dominant selectablemarker, the bacterial aadA gene encoding the spectinomycin-detoxifyingenzyme aminoglycoside-3′-adenyltransferase (Svab, Z., and Maliga, P.(1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this markerhad been used successfully for high-frequency transformation of theplastid genome of the green alga Chlamydomonas reinhardtii(Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19: 4083-4089). Otherselectable markers useful for plastid transformation are known in theart and encompassed within the scope of the invention. Typically,approximately 15-20 cell division cycles following transformation arerequired to reach a homoplastidic state. Plastid expression, in whichgenes are inserted by homologous recombination into all of the severalthousand copies of the circular plastid genome present in each plantcell, takes advantage of the enormous copy number advantage overnuclear-expressed genes to permit expression levels that can readilyexceed 10% of the total soluble plant protein. In a preferredembodiment, a nucleotide sequence of the present invention is insertedinto a plastid targeting vector and transformed into the plastid genomeof a desired plant host. Plants homoplastic for plastid genomescontaining a nucleotide sequence of the present invention are obtained,and are preferentially capable of high expression of the nucleotidesequence.

Formulation of Insecticidal Compositions

[0097] The invention also includes compositions comprising at least oneof the insecticidal toxins of the present invention. In order toeffectively control insect pests such compositions preferably containsufficient amounts of toxin. Such amounts vary depending on the crop tobe protected, on the particular pest to be targeted, and on theenvironmental conditions, such as humidity, temperature or type of soil.In a preferred embodiment, compositions comprising the insecticidaltoxins comprise host cells expressing the toxins without additionalpurification. In another preferred embodiment, the cells expressing theinsecticidal toxins are lyophilized prior to their use as aninsecticidal agent. In another embodiment, the insecticidal toxins areengineered to be secreted from the host cells. In cases wherepurification of the toxins from the host cells in which they areexpressed is desired, various degrees of purification of theinsecticidal toxins are reached.

[0098] The present invention further embraces the preparation ofcompositions comprising at least one insecticidal toxin of the presentinvention, which is homogeneously mixed with one or more compounds orgroups of compounds described herein. The present invention also relatesto methods of treating plants, which comprise application of theinsecticidal toxins or compositions containing the insecticidal toxins,to plants. The insecticidal toxins can be applied to the crop area inthe form of compositions or plant to be treated, simultaneously or insuccession, with further compounds. These compounds can be bothfertilizers or micronutrient donors or other preparations that influenceplant growth. They can also be selective herbicides, insecticides,fungicides, bactericides, nematicides, molluscicides or mixtures ofseveral of these preparations, if desired together with furthercarriers, surfactants or application-promoting adjuvants customarilyemployed in the art of formulation. Suitable carriers and adjuvants canbe solid or liquid and correspond to the substances ordinarily employedin formulation technology, e.g. natural or regenerated mineralsubstances, solvents, dispersants, wetting agents, tackifiers, bindersor fertilizers.

[0099] A preferred method of applying insecticidal toxins of the presentinvention is by spraying to the environment hosting the insect pest likethe soil, water, or foliage of plants. The number of applications andthe rate of application depend on the type and intensity of infestationby the insect pest. The insecticidal toxins can also penetrate the plantthrough the roots via the soil (systemic action) by impregnating thelocus of the plant with a liquid composition, or by applying thecompounds in solid form to the soil, e.g. in granular form (soilapplication). The insecticidal toxins may also be applied to seeds(coating) by impregnating the seeds either with a liquid formulationcontaining insecticidal toxins, or coating them with a solidformulation. In special cases, further types of application are alsopossible, for example, selective treatment of the plant stems or buds.The insecticidal toxins can also be provided as bait located above orbelow the ground.

[0100] The insecticidal toxins are used in unmodified form or,preferably, together with the adjuvants conventionally employed in theart of formulation, and are therefore formulated in known manner toemulsifiable concentrates, coatable pastes, directly sprayable ordilutable solutions, dilute emulsions, wettable powders, solublepowders, dusts, granulates, and also encapsulations, for example, inpolymer substances. Like the nature of the compositions, the methods ofapplication, such as spraying, atomizing, dusting, scattering orpouring, are chosen in accordance with the intended objectives and theprevailing circumstances.

[0101] The formulations, compositions or preparations containing theinsecticidal toxins and, where appropriate, a solid or liquid adjuvant,are prepared in known manner, for example by homogeneously mixing and/orgrinding the insecticidal toxins with extenders, for example solvents,solid carriers and, where appropriate, surface-active compounds(surfactants).

[0102] Suitable solvents include aromatic hydrocarbons, preferably thefractions having 8 to 12 carbon atoms, for example, xylene mixtures orsubstituted naphthalenes, phthalates such as dibutyl phthalate ordioctyl phthalate, aliphatic hydrocarbons such as cyclohexane orparaffins, alcohols and glycols and their ethers and esters, such asethanol, ethylene glycol monomethyl or monoethyl ether, ketones such ascyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone,dimethyl sulfoxide or dimethyl formamide, as well as epoxidizedvegetable oils such as epoxidized coconut oil or soybean oil or water.

[0103] The solid carriers used e.g. for dusts and dispersible powders,are normally natural mineral fillers such as calcite, talcum, kaolin,montmorillonite or attapulgite. In order to improve the physicalproperties it is also possible to add highly dispersed silicic acid orhighly dispersed absorbent polymers. Suitable granulated adsorptivecarriers are porous types, for example pumice, broken brick, sepioliteor bentonite; and suitable nonsorbent carriers are materials such ascalcite or sand. In addition, a great number of pregranulated materialsof inorganic or organic nature can be used, e.g. especially dolomite orpulverized plant residues.

[0104] Suitable surface-active compounds are nonionic, cationic and/oranionic surfactants having good emulsifying, dispersing and wettingproperties. The term “surfactants” will also be understood as comprisingmixtures of surfactants. Suitable anionic surfactants can be bothwater-soluble soaps and water-soluble synthetic surface-activecompounds.

[0105] Suitable soaps are the alkali metal salts, alkaline earth metalsalts or unsubstituted or substituted ammonium salts of higher fattyacids (chains of 10 to 22 carbon atoms), for example the sodium orpotassium salts of oleic or stearic acid, or of natural fatty acidmixtures which can be obtained for example from coconut oil or tallowoil. The fatty acid methylfuran salts may also be used.

[0106] More frequently, however, so-called synthetic surfactants areused, especially fatty sulfonates, fatty sulfates, sulfonatedbenzimidazole derivatives or alkylarylsulfonates.

[0107] The fatty sulfonates or sulfates are usually in the form ofalkali metal salts, alkaline earth metal salts or unsubstituted orsubstituted ammonium salts and have a 8 to 22 carbon alkyl radical whichalso includes the alkyl moiety of alkyl radicals, for example, thesodium or calcium salt of lignonsulfonic acid, of dodecylsulfate or of amixture of fatty alcohol sulfates obtained from natural fatty acids.These compounds also comprise the salts of sulfuric acid esters andsulfonic acids of fatty alcohol/ethylene oxide adducts. The sulfonatedbenzimidazole derivatives preferably contain 2 sulfonic acid groups andone fatty acid radical containing 8 to 22 carbon atoms. Examples ofalkylarylsulfonates are the sodium, calcium or triethanolamine salts ofdodecylbenzenesulfonic acid, dibutyinapthalenesulfonic acid, or of anaphthalenesulfonic acid/formaldehyde condensation product. Alsosuitable are corresponding phosphates, e.g. salts of the phosphoric acidester of an adduct of p-nonylphenol with 4 to 14 moles of ethyleneoxide.

[0108] Non-ionic surfactants are preferably polyglycol ether derivativesof aliphatic or cycloaliphatic alcohols, or saturated or unsaturatedfatty acids and alkylphenols, said derivatives containing 3 to 30 glycolether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbonmoiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.

[0109] Further suitable non-ionic surfactants are the water-solubleadducts of polyethylene oxide with polypropylene glycol, ethylenediaminepropylene glycol and alkylpolypropylene glycol containing 1 to 10 carbonatoms in the alkyl chain, which adducts contain 20 to 250 ethyleneglycol ether groups and 10 to 100 propylene glycol ether groups. Thesecompounds usually contain 1 to 5 ethylene glycol units per propyleneglycol unit.

[0110] Representative examples of non-ionic surfactants arenonylphenolpolyethoxyethanols, castor oil polyglycol ethers,polypropylene/polyethylene oxide adducts,tributylphenoxypolyethoxyethanol, polyethylene glycol andoctylphenoxyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitanand polyoxyethylene sorbitan trioleate are also suitable non-ionicsurfactants.

[0111] Cationic surfactants are preferably quaternary ammonium saltswhich have, as N-substituent, at least one C8-C22 alkyl radical and, asfurther substituents, lower unsubstituted or halogenated alkyl, benzylor lower hydroxyalkyl radicals. The salts are preferably in the form ofhalides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammoniumchloride or benzyldi(2-chloroethyl)ethylammonium bromide.

[0112] The surfactants customarily employed in the art of formulationare described, for example, in “McCutcheon's Detergents and EmulsifiersAnnual,” MC Publishing Corp. Ringwood, N.J., 1979, and Sisely and Wood,“Encyclopedia of Surface Active Agents,” Chemical Publishing Co., Inc.New York, 1980.

EXAMPLES

[0113] The invention will be further described by reference to thefollowing detailed examples. These examples are provided for purposes ofillustration only, and are not intended to be limiting unless otherwisespecified. Standard recombinant DNA and molecular cloning techniquesused here are well known in the art and are described by Ausubel (ed.),Current Protocols in Molecular Biology, John Wiley and Sons, Inc.(1994); T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor,N.Y. (1989); and by T. J. Silhavy, M. L. Berman, and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984).

A. Isolation of Nucleotide Sequences from Bacillus thuringiensis ssp.finitimus Whose Expression Results in Novel Delta-Endotoxins Example 1Bacterial Strain

[0114] Strain B-1166 VKPM Bacillus thuringiensis ssp. finitimus (BTfinitimus 1166) is used (Revina L. P., Zalunin I. A., Kriger I. V.,Tulina N. M., Wojciechowska J. A. Levitin E. I., and Chestukhina G. G.,Biokhimia (in press), incorporated herein by reference). This strain maybe purchased from VKPM (Russian State Collection of IndustrialMicroorganisms). This strain was deposited by M. Lecadet from PasteurInstitute (Paris, France) in 1985.

Example 2 Antibodies and Protein Assay

[0115] Rabbit antiserum is raised against a mixture of BT finitimus 1166true toxins obtained by chymotrypsin processing of parasporal inclusions(Revina et al., Biokhimia (in press)). The antiserum is pre-exhaustedwith crude extract of E. coli NM522 and purified by affinitychromatography on immobilized BT finitimus 1166 delta-endotoxin mix asdescribed in Sambrook, J., Fritsch, E., Maniatis, T. (1989).

Example 3 Genomic Bank Construction and Screening

[0116] BT finitimus 1166 total DNA is isolated as described by Delecluseet al., J. Bacteriol. 173: 3374-3381 (1991) and partially digested withSau3A. DNA fragments exceeding 5 kb in size are recovered from anagarose gel and ligated into the BamHI-linearized pUK21 vector (Vieira,J. and Messing, J., Gene 100: 189-194 (1991)) treated with CIAP. E. coliNM522 is transformed with ligation mix and plated on LB agar mediumsupplemented with kanamycin and IPTG. About 3000 clones are screenedwith the rabbit antiserum and two colonies are selected for furtheranalysis.

Example 4 Sequencing

[0117] Sequencing is performed using Taq DNA polymerase modification ofSanger method (Sambrook, J., Fritsch, E., Maniatis, T. (1989)) with aset of overlapping subclones ensuring a complete sequencing of bothstrands of the cloned fragments.

Example 5 Expression of the Cloned Genes

[0118] The cloned genes are expressed in E. coli NM522 as a host instandard LB broth supplemented with 20 mkg/ml kanamycin and 0.1 mM IPTG.Cell cultures are grown overnight at 30° C. Western and southern blotanalyses are performed following standard protocols.

Example 6 Cloning and Sequence Analysis

[0119] Two independent clones pF1 and pF2 are selected by screening ofthe genomic bank of BT finitimus 1166 with antiserum. Sequence analysisof the 6930 bp-fragment in pF1 (SEQ ID NO: 1) and 4896 bp-fragment inpF2 (SEQ ID NO: 3) reveals a single long open reading frame (ORF) ineach of them. Both deduced amino acid sequences (SEQ ID NOs: 2 and 4,respectively) are found most homologous to those of the Cry1-Cry9 group(35-42% identity). However, they are different enough to warrant two newprimary ranks as cry26Aa1 (pF1 insertion) and cry28Aa1 (pF2 insertion).Cry26Aa1 (SEQ ID NO: 2) and Cry28Aa1 (SEQ ID NO: 4) are more similar inC-terminal rather than in N-terminal moiety (64% and 36% identity,respectively).

[0120] A sequence of 7 amino acid residues determined in N-terminus ofmajor chymotrypsin processed Cry protein of BT finitimus 1166corresponds to that occurring within the deduced amino acid sequence ofthe Cry26Aa1 (SEQ ID NO: 2). This protein is equally distributed betweenboth types of BT finitimus 1166 parasporal inclusions, inspore-associated and in free ones (Revina et al., Biokhimia (in press)).

[0121] The cloned fragment harboring the cry26Aa1 gene is lacking in BtIand BtII or any other conserved BT promoter sequences. However,efficient production of the Cry26Aa1 protein implies efficienttranscription of cry26Aa1 gene in the BT finitimus 1166 strain. Near thecry26Aa1 ORF, a ribosome-binding sequence (GGAGG) is found.

[0122] The cloned fragment harbouring the cry28Aa1 gene is also lackingin BtI and BtII promoter sequences. A putative vegetative promotersequence TTGCAA(N)₁₅TAAGCC (SEQ ID NO: 5) similar to that of cry3Aa islocated 280 bp upstream of the cry28Aa1 ORF. Near the cry28Aa1a ORF, aputative ribosome-binding sequence AAAGG complementary to the3′-terminal region of 16S rRNA is found.

[0123] Recombinant plasmids pF1 and pF2 provide efficient expression ofCry26Aa1 and Cry28Aa1 in E. coli cells. Cry26Aa1 is also expressed intruncated form of 506 N-terminal amino acid residues (amino acids 1-506of SEQ ID NO: 4). Molecular weight of the products is estimated as 130kD for pF2, 125 kD for pF2, and 65 kD for truncated pF1.

Example 7 Examination of Regions Flanking the cry26Aa1 Gene in the BTfinitimus Genome

[0124] The lack of conventional BT promoters suggests a number ofCry26Aa1 alleles differing in genomic surrounding and allowingdifferential control of expression. A restriction map of the cry26Aa1upstream flanking region in the BT finitimus 1166 genome is studied bySouthern hybridization. A PauI-PstI DNA fragment containing the5′-terminus of the cry26Aa1 gene is used as a probe. Total BT finitimus1166 genomic DNA samples are digested with seven pairs of restrictionendonucleases; only one hybridized fragment is observed by examinationof the region within 7.5 kb from the translation start in each case. Thedownstream flanking region also has a unique restriction map. Thissuggests that the protein Cry26Aa1 in both spore-associated and freetypes of crystals is synthesized under control of one and the samegenomic locus. Southern hybridization also demonstrates uniquesurroundings in BT finitimus genome for at least 3 kb both upstream anddownstream of the cry28Aa1 ORF.

B. Expression of the Nucleic Acid Sequences of the Invention inHeterologous Microbial Hosts

[0125] Microorganisms which are suitable for the heterologous expressionof the nucleotide sequences of the invention are all microorganismswhich are capable of colonizing plants or the rhizosphere. As such theywill be brought into contact with insect pests. These includegram-negative microorganisms such as Pseudomonas, Enterobacter andSerratia, the gram-positive microorganism Bacillus and the fungiTrichoderma, Gliocladium, and Saccharomyces cerevisiae. Particularlypreferred heterologous hosts are Pseudomonas fluorescens, Pseudomonasputida, Pseudomonas cepacia, Pseudomonas aureofaciens, Pseudomonasaurantiaca, Enterobacter cloacae, Serratia marcescens, Bacillussubtilis, Bacillus cereus, Trichoderma viride, Trichoderma harzianum,Gliocladium virens, and Saccharomyces cerevisiae.

Example 8 Expression of the Nucleotide Sequences in E. coli and OtherGram-Negative Bacteria

[0126] Many genes have been expressed in gram-negative bacteria in aheterologous manner. Expression vector pKK223-3 (Pharmacia catalogue#27-4935-01) allows expression in E. coli. This vector has a strong tacpromoter (Brosius, J. et al., Proc. Natl. Acad. Sci. USA 81) regulatedby the lac repressor and induced by IPTG. A number of other expressionsystems have been developed for use in E. coli. The thermoinducibleexpression vector pP_(L) (Pharmacia #27-4946-01) uses a tightlyregulated bacteriophage λ promoter which allows for high levelexpression of proteins. The lac promoter provides another means ofexpression but the promoter is not expressed at such high levels as thetac promoter. With the addition of broad host range replicons to some ofthese expression system vectors, expression of the nucleotide sequencein closely related gram negative-bacteria such as Pseudomonas,Enterobacter, Serratia and Erwinia is possible. For example, pLRKD211(Kaiser & Kroos, Proc. Natl. Acad. Sci. USA 81: 5816-5820 (1984))contains the broad host range replicon ori T which allows replication inmany gram-negative bacteria.

[0127] In E. coli, induction by IPTG is required for expression of thetac (i.e. trp-lac) promoter. When this same promoter (e.g. on wide-hostrange plasmid pLRKD211) is introduced into Pseudomonas it isconstitutively active without induction by IPTG. This trp-lac promotercan be placed in front of any gene or operon of interest for expressionin Pseudomonas or any other closely related bacterium for the purposesof the constitutive expression of such a gene. Thus, a nucleotidesequence whose expression results in an insecticidal toxin can thereforebe placed behind a strong constitutive promoter, transferred to abacterium which has plant or rhizosphere colonizing properties turningthis organism to an insecticidal agent. Other possible promoters can beused for the constitutive expression of the nucleotide sequence ingram-negative bacteria. These include, for example, the promoter fromthe Pseudomonas regulatory genes gafA and lemA (WO 94/01561) and thePseudomonas savastanoi IAA operon promoter (Gaffney et al., J.Bacteriol. 172: 5593-5601 (1990).

Example 9 Expression of the Nucleotide Sequences in Gram-PositiveBacteria

[0128] Heterologous expression of the nucleotides sequence ingram-positive bacteria is another means of producing the insecticidaltoxins. Expression systems for Bacillus and Streptomyces are the bestcharacterized. The promoter for the erythromycin resistance gene (ermR)from Streptococcus pneumoniae has been shown to be active ingram-positive aerobes and anaerobes and also in E. coli (Trieu-Cuot etal., Nucl Acids Res 18: 3660 (1990)). A further antibiotic resistancepromoter from the thiostreptone gene has been used in Streptomycescloning vectors (Bibb, Mol Gen Genet 199: 26-36 (1985)). The shuttlevector pHT3101 is also appropriate for expression in Bacillus (Lereclus,FEMS Microbiol Lett 60: 211-218 (1989)). A significant advantage of thisapproach is that many gram-positive bacteria produce spores which can beused in formulations that produce insecticidal agents with a longershelf life. Bacillus and Streptomyces species are aggressive colonizersof soils

Example 10 Expression of the Nucleotide Sequences in Fungi

[0129]Trichoderma harzianum and Gliocladium virens have been shown toprovide varying levels of biocontrol in the field (U.S. Pat. Nos.5,165,928 and 4,996,157, both to Cornell Research Foundation). Anucleotide sequence whose expression results in an insecticidal toxincould be expressed in such a fungus. This could be accomplished by anumber of ways which are well known in the art. One isprotoplast-mediated transformation of the fungus by PEG orelectroporation-mediated techniques. Alternatively, particle bombardmentcan be used to transform protoplasts or other fungal cells with theability to develop into regenerated mature structures. The vectorpAN7-1, originally developed for Aspergillus transformation and now usedwidely for fungal transformation (Curragh et al., Mycol. Res. 97(3):313-317 (1992); Tooley et al., Curr. Genet. 21: 55-60 (1992); Punt etal., Gene 56: 117-124 (1987)) is engineered to contain the nucleotidesequence. This plasmid contains the E. coli the hygromycin B resistancegene flanked by the Aspergillus nidulans gpd promoter and the trpCterminator (Punt et al., Gene 56: 117-124 (1987)). In a preferredembodiment, the nucleic acid sequences of the invention are expressed inthe yeast Saccharomyces cerevisiae.

C. Formulation of the Insecticidal Toxin

[0130] Insecticidal formulations are made using active ingredients whichcomprise either the isolated toxin or alternatively suspensions orconcentrates of cells which produce it and which are described in theexamples above. For example, E. coli cells expressing an insecticidaltoxin of the invention may be used for the control of the insect pests.Formulations are made in liquid or solid form and are described below.

Example 11 Liquid Formulation of Insecticidal Compositions

[0131] In the following examples, percentages of composition are givenby weight: 1. Emulsifiable concentrates: a b c Active ingredient 20% 40%50% Calcium dodecylbenzenesulfonate 5% 8% 6% Castor oil polyethleneglycol 5% — — ether (36 moles of ethylene oxide) Tributylphenolpolyethylene glyco — 12% 4% ether (30 moles of ethylene oxide)Cyclohexanone — 15% 20% Xylene mixture 70% 25% 20%

[0132] Emulsions of any required concentration can be produced from suchconcentrates by dilution with water. 2. Solutions: a b c d Activeingredient 80% 10% 5% 95% Ethylene glycol monomethyl ether 20% — — —Polyethylene glycol 400 — 70% — — N-methyl-2-pyrrolidone — 20% — —Epoxidised coconut oil — — 1% 5% Petroleum distillate — — 94% — (boilingrange 160-190° C.)

[0133] These solutions are suitable for application in the form ofmicrodrops. 3. Granulates: a b Active ingredient 5% 10% Kaolin 94% —Highly dispersed silicic acid 1% — Attapulgit — 90%

[0134] The active ingredient is dissolved in methylene chloride, thesolution is sprayed onto the carrier, and the solvent is subsequentlyevaporated off in vacuo. 4. Dusts: a b Active ingredient 2% 5% Highlydispersed silicic acid 1% 5% Talcum 97% — Kaolin — 90%

[0135] Ready-to-use dusts are obtained by intimately mixing the carrierswith the active ingredient.

Example 12 Solid Formulation of Insecticidal Compositions

[0136] In the following examples, percentages of compositions are byweight. 1. Wettable powders: a b c Active ingredient 20% 60% 75% Sodiumlignosulfonate 5% 5% — Sodium lauryl sulfate 3% — 5% Sodiumdiisobutylnaphthalene sulfonate — 6% 10% Octylphenol polyethylene glycolether — 2% — (7-8 moles of ethylene oxide) Highly dispersed silicic acid5% 27% 10% Kaolin 67% — —

[0137] The active ingredient is thoroughly mixed with the adjuvants andthe mixture is thoroughly ground in a suitable mill, affording wettablepowders which can be diluted with water to give suspensions of thedesired concentrations. 2. Emulsifiable concentrate: Active ingredient10% Octylphenol polyethylene glycol ether 3% (4-5 moles of ethyleneoxide) Calcium dodecylbenzenesulfonate 3% Castor oil polyglycol ether 4%(36 moles of ethylene oxide) Cyclohexanone 30% Xylene mixture 50%

[0138] Emulsions of any required concentration can be obtained from thisconcentrate by dilution with water. 3. Dusts: a b Active ingredient 5%8% Talcum 95% — Kaolin — 92%

[0139] Ready-to-use dusts are obtained by mixing the active ingredientwith the carriers, and grinding the mixture in a suitable mill. 4.Extruder granulate: Active ingredient 10% Sodium lignosulfonate 2%Carboxymethylcellulose 1% Kaolin 87%

[0140] The active ingredient is mixed and ground with the adjuvants, andthe mixture is subsequently moistened with water. The mixture isextruded and then dried in a stream of air. 5. Coated granulate: Activeingredient 3% Polyethylene glycol 200 3% Kaolin 94%

[0141] The finely ground active ingredient is uniformly applied, in amixer, to the kaolin moistened with polyethylene glycol. Non-dustycoated granulates are obtained in this manner. 6. Suspensionconcentrate: Active ingredient 40% Ethylene glycol 10% Nonylphenolpolyethylene glycol 6% (15 moles of ethylene oxide) Sodiumlignosulfonate 10% Carboxymethylcellulose 1% 37% aqueous formaldehydesolution 0.2% Silicone oil in 75% aqueous emulsion 0.8% Water 32%

[0142] The finely ground active ingredient is intimately mixed with theadjuvants, giving a suspension concentrate from which suspensions of anydesire concentration can be obtained by dilution with water.

[0143] The insecticidal formulations described above are applied to theplants according to methods well known in the art, in such amounts thatthe insect pests are controlled by the insecticidal toxin.

D. Expression of the Nucleotide Sequences in Transgenic Plants

[0144] The nucleic acid sequences described in this application can beincorporated into plant cells using conventional recombinant DNAtechnology. Generally, this involves inserting a coding sequence of theinvention into an expression system to which the coding sequence isheterologous (i.e., not normally present) using standard cloningprocedures known in the art. The vector contains the necessary elementsfor the transcription and translation of the inserted protein-codingsequences. A large number of vector systems known in the art can beused, such as plasmids, bacteriophage viruses and other modifiedviruses. Suitable vectors include, but are not limited to, viral vectorssuch as lambda vector systems λgtI1, λgtI0 and Charon 4; plasmid vectorssuch as pBI121, pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8,pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101, pCDNAII; and othersimilar systems. The components of the expression system may also bemodified to increase expression. For example, truncated sequences,nucleotide substitutions or other modifications may be employed. Theexpression systems described herein can be used to transform virtuallyany crop plant cell under suitable conditions. Transformed cells can beregenerated into whole plants such that the nucleotide sequence of theinvention confer insect resistance to the transgenic plants.

Example 13 Modification of Coding Sequences and Adjacent Sequences

[0145] The nucleotide sequences described in this application can bemodified for expression in transgenic plant hosts. A host plantexpressing the nucleotide sequences and which produces the insecticidaltoxins in its cells has enhanced resistance to insect attack and is thusbetter equipped to withstand crop losses associated with such attack.

[0146] The transgenic expression in plants of genes derived frommicrobial sources may require the modification of those genes to achieveand optimize their expression in plants. In particular, bacterial ORFswhich encode separate enzymes but which are encoded by the sametranscript in the native microbe are best expressed in plants onseparate transcripts. To achieve this, each microbial ORF is isolatedindividually and cloned within a cassette which provides a plantpromoter sequence at the 5′ end of the ORF and a plant transcriptionalterminator at the 3′ end of the ORF. The isolated ORF sequencepreferably includes the initiating ATG codon and the terminating STOPcodon but may include additional sequence beyond the initiating ATG andthe STOP codon. In addition, the ORF may be truncated, but still retainthe required activity; for particularly long ORFs, truncated versionswhich retain activity may be preferable for expression in transgenicorganisms. By “plant promoter” and “plant transcriptional terminator” itis intended to mean promoters and transcriptional terminators whichoperate within plant cells. This includes promoters and transcriptionterminators which may be derived from non-plant sources such as viruses(an example is the Cauliflower Mosaic Virus).

[0147] In some cases, modification to the ORF coding sequences andadjacent sequence is not required. It is sufficient to isolate afragment containing the ORF of interest and to insert it downstream of aplant promoter. For example, Gaffney et al. (Science 261: 754-756(1993)) have expressed the Pseudomonas nahG gene in transgenic plantsunder the control of the CaMV 35S promoter and the CaMV tmI terminatorsuccessfully without modification of the coding sequence and with x bpof the Pseudomonas gene upstream of the ATG still attached, and y bpdownstream of the STOP codon still attached to the nahG ORF. Preferablyas little adjacent microbial sequence should be left attached upstreamof the ATG and downstream of the STOP codon. In practice, suchconstruction may depend on the availability of restriction sites.

[0148] In other cases, the expression of genes derived from microbialsources may provide problems in expression. These problems have beenwell characterized in the art and are particularly common with genesderived from certain sources such as Bacillus. These problems may applyto the nucleotide sequence of this invention and the modification ofthese genes can be undertaken using techniques now well known in theart. The following problems may be encountered:

[0149] 1. Codon Usage.

[0150] The preferred codon usage in plants differs from the preferredcodon usage in certain microorganisms. Comparison of the usage of codonswithin a cloned microbial ORF to usage in plant genes (and in particulargenes from the target plant) will enable an identification of the codonswithin the ORF which should preferably be changed. Typically plantevolution has tended towards a strong preference of the nucleotides Cand G in the third base position of monocotyledons, whereas dicotyledonsoften use the nucleotides A or T at this position. By modifying a geneto incorporate preferred codon usage for a particular target transgenicspecies, many of the problems described below for GC/AT content andillegitimate splicing will be overcome.

[0151] 2. GC/AT Content.

[0152] Plant genes typically have a GC content of more than 35%. ORFsequences which are rich in A and T nucleotides can cause severalproblems in plants. Firstly, motifs of ATTTA are believed to causedestabilization of messages and are found at the 3′ end of manyshort-lived mRNAs. Secondly, the occurrence of polyadenylation signalssuch as AATAAA at inappropriate positions within the message is believedto cause premature truncation of transcription. In addition,monocotyledons may recognize AT-rich sequences as splice sites (seebelow).

[0153] 3. Sequences Adjacent to the Initiating Methionine.

[0154] Plants differ from microorganisms in that their messages do notpossess a defined ribosome binding site. Rather, it is believed thatribosomes attach to the 5′ end of the message and scan for the firstavailable ATG at which to start translation. Nevertheless, it isbelieved that there is a preference for certain nucleotides adjacent tothe ATG and that expression of microbial genes can be enhanced by theinclusion of a eukaryotic consensus translation initiator at the ATG.Clontech (1993/1994 catalog, page 210, incorporated herein by reference)have suggested one sequence as a consensus translation initiator for theexpression of the E. coli uidA gene in plants. Further, Joshi (NAR 15:6643-6653 (1987), incorporated herein by reference) has compared manyplant sequences adjacent to the ATG and suggests another consensussequence. In situations where difficulties are encountered in theexpression of microbial ORFs in plants, inclusion of one of thesesequences at the initiating ATG may improve translation. In such casesthe last three nucleotides of the consensus may not be appropriate forinclusion in the modified sequence due to their modification of thesecond AA residue. Preferred sequences adjacent to the initiatingmethionine may differ between different plant species. A survey of 14maize genes located in the GenBank database provided the followingresults: Position Before the Initiating ATG in 14 Maize Genes: −10 −9 −8−7 −6 −5 −4 −3 −2 −1 C 3 8 4 6 2 5 6 0 10 7 T 3 0 3 4 3 2 1 1 1 0 A 2 31 4 3 2 3 7 2 3 G 6 3 6 0 6 5 4 6 1 5

[0155] This analysis can be done for the desired plant species intowhich the nucleotide sequence is being incorporated, and the sequenceadjacent to the ATG modified to incorporate the preferred nucleotides.

[0156] 4. Removal of Illegitimate Splice Sites.

[0157] Genes cloned from non-plant sources and not optimized forexpression in plants may also contain motifs which may be recognized inplants as 5′ or 3′ splice sites, and be cleaved, thus generatingtruncated or deleted messages. These sites can be removed using thetechniques well known in the art.

[0158] Techniques for the modification of coding sequences and adjacentsequences are well known in the art. In cases where the initialexpression of a microbial ORF is low and it is deemed appropriate tomake alterations to the sequence as described above, then theconstruction of synthetic genes can be accomplished according to methodswell known in the art. These are, for example, described in thepublished patent disclosures EP 0 385 962 (to Monsanto), EP 0 359 472(to Lubrizol) and WO 93/07278 (to Ciba-Geigy), all of which areincorporated herein by reference. In most cases it is preferable toassay the expression of gene constructions using transient assayprotocols (which are well known in the art) prior to their transfer totransgenic plants.

Example 14 Construction of Plant Expression Cassettes

[0159] Coding sequences intended for expression in transgenic plants arefirst assembled in expression cassettes behind a suitable promoterexpressible in plants. The expression cassettes may also comprise anyfurther sequences required or selected for the expression of thetransgene. Such sequences include, but are not restricted to,transcription terminators, extraneous sequences to enhance expressionsuch as introns, vital sequences, and sequences intended for thetargeting of the gene product to specific organelles and cellcompartments. These expression cassettes can then be easily transferredto the plant transformation vectors described below. The following is adescription of various components of typical expression cassettes.

[0160] 1. Promoters

[0161] The selection of the promoter used in expression cassettes willdetermine the spatial and temporal expression pattern of the transgenein the transgenic plant. Selected promoters will express transgenes inspecific cell types (such as leaf epidermal cells, mesophyll cells, rootcortex cells) or in specific tissues or organs (roots, leaves orflowers, for example) and the selection will reflect the desiredlocation of accumulation of the gene product. Alternatively, theselected promoter may drive expression of the gene under variousinducing conditions. Promoters vary in their strength, i.e., ability topromote transcription. Depending upon the host cell system utilized, anyone of a number of suitable promoters can be used, including the gene'snative promoter. The following are non-limiting examples of promotersthat may be used in expression cassettes.

[0162] a. Constitutive Expression, the Ubiquitin Promoter:

[0163] Ubiquitin is a gene product known to accumulate in many celltypes and its promoter has been cloned from several species for use intransgenic plants (e.g. sunflower—Binet et al. Plant Science 79: 87-94(1991); maize—Christensen et al. Plant Molec. Biol. 12: 619-632 (1989);and Arabidopsis—Norris et al., Plant Mol. Biol. 21:895-906 (1993)). Themaize ubiquitin promoter has been developed in transgenic monocotsystems and its sequence and vectors constructed for monocottransformation are disclosed in the patent publication EP 0 342 926 (toLubrizol) which is herein incorporated by reference. Taylor et al.(Plant Cell Rep. 12: 491-495 (1993)) describe a vector (pAHC25) thatcomprises the maize ubiquitin promoter and first intron and its highactivity in cell suspensions of numerous monocotyledons when introducedvia microprojectile bombardment. The Arabidopsis ubiquitin promoter isideal for use with the nucleotide sequences of the present invention.The ubiquitin promoter is suitable for gene expression in transgenicplants, both monocotyledons and dicotyledons. Suitable vectors arederivatives of pAHC25 or any of the transformation vectors described inthis application, modified by the introduction of the appropriateubiquitin promoter and/or intron sequences.

[0164] b. Constitutive Expression, the CaMV 35S Promoter:

[0165] Construction of the plasmid pCGN1761 is described in thepublished patent application EP 0 392 225 (Example 23), which is herebyincorporated by reference. pCGN1761 contains the “double” CaMV 35Spromoter and the tmI transcriptional terminator with a unique EcoRI sitebetween the promoter and the terminator and has a pUC-type backbone. Aderivative of pCGN1761 is constructed which has a modified polylinkerwhich includes NotI and XhoI sites in addition to the existing EcoRIsite. This derivative is designated pCGN1761 ENX. pCGN1761ENX is usefulfor the cloning of cDNA sequences or coding sequences (includingmicrobial ORF sequences) within its polylinker for the purpose of theirexpression under the control of the 35S promoter in transgenic plants.The entire 35S promoter-coding sequence-tmI terminator cassette of sucha construction can be excised by HindIII, SphI, SaII, and XbaI sites 5′to the promoter and XbaI, BamHI and BgII sites 3′ to the terminator fortransfer to transformation vectors such as those described below.Furthermore, the double 35S promoter fragment can be removed by 5′excision with HindIII, SphI, SaII, XbaI, or PstI, and 3′ excision withany of the polylinker restriction sites (EcoRI, NotI or XhoI) forreplacement with another promoter. If desired, modifications around thecloning sites can be made by the introduction of sequences that mayenhance translation. This is particularly useful when overexpression isdesired. For example, pCGN1761ENX may be modified by optimization of thetranslational initiation site as described in Example 37 of U.S. Pat.No. 5,639,949, incorporated herein by reference.

[0166] c. Constitutive Expression, the Actin Promoter:

[0167] Several isoforms of actin are known to be expressed in most celltypes and consequently the actin promoter is a good choice for aconstitutive promoter. In particular, the promoter from the rice ActIgene has been cloned and characterized (McElroy et al. Plant Cell 2:163-171 (1990)). A 1.3 kb fragment of the promoter was found to containall the regulatory elements required for expression in rice protoplasts.Furthermore, numerous expression vectors based on the ActI promoter havebeen constructed specifically for use in monocotyledons (McElroy et al.Mol. Gen. Genet. 231: 150-160 (1991)). These incorporate the ActI-intron1, AdhI 5′ flanking sequence and AdhI-intron 1 (from the maize alcoholdehydrogenase gene) and sequence from the CaMV 35S promoter. Vectorsshowing highest expression were fusions of 35S and ActI intron or theActI 5′ flanking sequence and the ActI intron. Optimization of sequencesaround the initiating ATG (of the GUS reporter gene) also enhancedexpression. The promoter expression cassettes described by McElroy etal. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified forgene expression and are particularly suitable for use inmonocotyledonous hosts. For example, promoter-containing fragments isremoved from the McElroy constructions and used to replace the double35S promoter in pCGN1761ENX, which is then available for the insertionof specific gene sequences. The fusion genes thus constructed can thenbe transferred to appropriate transformation vectors. In a separatereport, the rice ActI promoter with its first intron has also been foundto direct high expression in cultured barley cells (Chibbar et al. PlantCell Rep. 12: 506-509 (1993)).

[0168] d. Inducible Expression, the PR-1 Promoter:

[0169] The double 35S promoter in pCGN1761ENX may be replaced with anyother promoter of choice that will result in suitably high expressionlevels. By way of example, one of the chemically regulatable promotersdescribed in U.S. Pat. No. 5,614,395 may replace the double 35Spromoter. The promoter of choice is preferably excised from its sourceby restriction enzymes, but can alternatively be PCR-amplified usingprimers that carry appropriate terminal restriction sites. ShouldPCR-amplification be undertaken, then the promoter should bere-sequenced to check for amplification errors after the cloning of theamplified promoter in the target vector. The chemically/pathogenregulatable tobacco PR-1a promoter is cleaved from plasmid pCIB1004 (forconstruction, see example 21 of EP 0 332 104, which is herebyincorporated by reference) and transferred to plasmid pCGN1761ENX (Ukneset al., 1992). pCIB1004 is cleaved with NcoI and the resultant 3′overhang of the linearized fragment is rendered blunt by treatment withT4 DNA polymerase. The fragment is then cleaved with HindIII and theresultant PR-1a promoter-containing fragment is gel purified and clonedinto pCGN1761ENX from which the double 35S promoter has been removed.This is done by cleavage with XhoI and blunting with T4 polymerase,followed by cleavage with HindIII and isolation of the largervector-terminator containing fragment into which the pCIB1004 promoterfragment is cloned. This generates a pCGN1761ENX derivative with thePR-1a promoter and the tmI terminator and an intervening polylinker withunique EcoRI and NotI sites. The selected coding sequence can beinserted into this vector, and the fusion products (i.e.promoter-gene-terminator) can subsequently be transferred to anyselected transformation vector, including those described infra. Variouschemical regulators may be employed to induce expression of the selectedcoding sequence in the plants transformed according to the presentinvention, including the benzothiadiazole, isonicotinic acid, andsalicylic acid compounds disclosed in U.S. Pat. Nos. 5,523,311 and5,614,395.

[0170] e. Inducible Expression, an Ethanol-Inducible Promoter:

[0171] A promoter inducible by certain alcohols or ketones, such asethanol, may also be used to confer inducible expression of a codingsequence of the present invention. Such a promoter is for example theaIcA gene promoter from Aspergillus nidulans (Caddick et al. (1998) Nat.Biotechnol 16:177-180). In A. nidulans, the aIcA gene encodes alcoholdehydrogenase 1, the expression of which is regulated by the AIcRtranscription factors in presence of the chemical inducer. For thepurposes of the present invention, the CAT coding sequences in plasmidpaIcA:CAT comprising a aIcA gene promoter sequence fused to a minimal35S promoter (Caddick et al. (1998) Nat. Biotechnol 16:177-180) arereplaced by a coding sequence of the present invention to form anexpression cassette having the coding sequence under the control of theaIcA gene promoter. This is carried out using methods well known in theart.

[0172] f. Inducible Expression, a Glucocorticoid-Inducible Promoter:

[0173] Induction of expression of a nucleic acid sequence of the presentinvention using systems based on steroid hormones is also contemplated.For example, a glucocorticoid-mediated induction system is used (Aoyamaand Chua (1997) The Plant Journal 11: 605-612) and gene expression isinduced by application of a glucocorticoid, for example a syntheticglucocorticoid, preferably dexamethasone, preferably at a concentrationranging from 0.1 mM to 1 mM, more preferably from 10 mM to 100 mM. Forthe purposes of the present invention, the luciferase gene sequences arereplaced by a nucleic acid sequence of the invention to form anexpression cassette having a nucleic acid sequence of the inventionunder the control of six copies of the GAL4 upstream activatingsequences fused to the 35S minimal promoter. This is carried out usingmethods well known in the art. The trans-acting factor comprises theGAL4 DNA-binding domain (Keegan et al. (1986) Science 231: 699-704)fused to the transactivating domain of the herpes viral protein VP16(Triezenberg et al. (1988) Genes Devel. 2: 718-729) fused to thehormone-binding domain of the rat glucocorticoid receptor (Picard et al.(1988) Cell 54: 1073-1080). The expression of the fusion protein iscontrolled by any promoter suitable for expression in plants known inthe art or described here. This expression cassette is also comprised inthe plant comprising a nucleic acid sequence of the invention fused tothe 6xGAL4/minimal promoter. Thus, tissue- or organ-specificity of thefusion protein is achieved leading to inducible tissue- ororgan-specificity of the insecticidal toxin.

[0174] g. Root Specific Expression:

[0175] Another pattern of gene expression is root expression. A suitableroot promoter is described by de Framond (FEBS 290: 103-106 (1991)) andalso in the published patent application EP 0 452 269, which is hereinincorporated by reference. This promoter is transferred to a suitablevector such as pCGN1761 ENX for the insertion of a selected gene andsubsequent transfer of the entire promoter-gene-terminator cassette to atransformation vector of interest.

[0176] h. Wound-Inducible Promoters:

[0177] Wound-inducible promoters may also be suitable for geneexpression. Numerous such promoters have been described (e.g. Xu et al.Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792(1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), Warner etal. Plant J. 3: 191-201 (1993)) and all are suitable for use with theinstant invention. Logemann et al. describe the 5′ upstream sequences ofthe dicotyledonous potato wunI gene. Xu et al. show that awound-inducible promoter from the dicotyledon potato (pin2) is active inthe monocotyledon rice. Further, Rohrmeier & Lehle describe the cloningof the maize WipI cDNA which is wound induced and which can be used toisolate the cognate promoter using standard techniques. Similar, Fireket al. and Warner et al. have described a wound-induced gene from themonocotyledon Asparagus officinalis, which is expressed at local woundand pathogen invasion sites. Using cloning techniques well known in theart, these promoters can be transferred to suitable vectors, fused tothe genes pertaining to this invention, and used to express these genesat the sites of plant wounding.

[0178] i. Pith-Preferred Expression:

[0179] Patent Application WO 93/07278, which is herein incorporated byreference, describes the isolation of the maize trpA gene, which ispreferentially expressed in pith cells. The gene sequence and promoterextending up to −1726 bp from the start of transcription are presented.Using standard molecular biological techniques, this promoter, or partsthereof, can be transferred to a vector such as pCGN1761 where it canreplace the 35S promoter and be used to drive the expression of aforeign gene in a pith-preferred manner. In fact, fragments containingthe pith-preferred promoter or parts thereof can be transferred to anyvector and modified for utility in transgenic plants.

[0180] j. Leaf-Specific Expression:

[0181] A maize gene encoding phosphoenol carboxylase (PEPC) has beendescribed by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)).Using standard molecular biological techniques the promoter for thisgene can be used to drive the expression of any gene in a leaf-specificmanner in transgenic plants.

[0182] k. Pollen-Specific Expression:

[0183] WO 93/07278 describes the isolation of the maizecalcium-dependent protein kinase (CDPK) gene which is expressed inpollen cells. The gene sequence and promoter extend up to 1400 bp fromthe start of transcription. Using standard molecular biologicaltechniques, this promoter or parts thereof, can be transferred to avector such as pCGN1761 where it can replace the 35S promoter and beused to drive the expression of a nucleic acid sequence of the inventionin a pollen-specific manner.

[0184] 2. Transcriptional Terminators

[0185] A variety of transcriptional terminators are available for use inexpression cassettes. These are responsible for the termination oftranscription beyond the transgene and its correct polyadenylation.Appropriate transcriptional terminators are those that are known tofunction in plants and include the CaMV 35S terminator, the tmIterminator, the nopaline synthase terminator and the pea rbcS E9terminator. These can be used in both monocotyledons and dicotyledons.In addition, a gene's native transcription terminator may be used.

[0186] 3. Sequences for the Enhancement or Regulation of Expression

[0187] Numerous sequences have been found to enhance gene expressionfrom within the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants.

[0188] Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, the introns of themaize AdhI gene have been found to significantly enhance the expressionof the wild-type gene under its cognate promoter when introduced intomaize cells. Intron 1 was found to be particularly effective andenhanced expression in fusion constructs with the chloramphenicolacetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200(1987)). In the same experimental system, the intron from the maizebronzel gene had a similar effect in enhancing expression. Intronsequences have been routinely incorporated into plant transformationvectors, typically within the non-translated leader.

[0189] A number of non-translated leader sequences derived from virusesare also known to enhance expression, and these are particularlyeffective in dicotyledonous cells. Specifically, leader sequences fromTobacco Mosaic Virus (TMV, the “W-sequence”), Maize Chlorotic MottleVirus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to beeffective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res.15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79(1990)).

[0190] 4. Targeting of the Gene Product Within the Cell

[0191] Various mechanisms for targeting gene products are known to existin plants and the sequences controlling the functioning of thesemechanisms have been characterized in some detail. For example, thetargeting of gene products to the chloroplast is controlled by a signalsequence found at the amino terminal end of various proteins which iscleaved during chloroplast import to yield the mature protein (e.g.Comai et al. J. Biol. Chem. 263: 15104-15109 (1988)). These signalsequences can be fused to heterologous gene products to effect theimport of heterologous products into the chloroplast (van den Broeck, etal. Nature 313: 358-363 (1985)). DNA encoding for appropriate signalsequences can be isolated from the 5′ end of the cDNAs encoding theRUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2protein and many other proteins which are known to be chloroplastlocalized. See also, the section entitled “Expression With ChloroplastTargeting” in Example 37 of U.S. Pat. No. 5,639,949.

[0192] Other gene products are localized to other organelles such as themitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol.13: 411-418 (1989)). The cDNAs encoding these products can also bemanipulated to effect the targeting of heterologous gene products tothese organelles. Examples of such sequences are the nuclear-encodedATPases and specific aspartate amino transferase isoforms formitochondria. Targeting cellular protein bodies has been described byRogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)).

[0193] In addition, sequences have been characterized which cause thetargeting of gene products to other cell compartments. Amino terminalsequences are responsible for targeting to the ER, the apoplast, andextracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2:769-783 (1990)). Additionally, amino terminal sequences in conjunctionwith carboxy terminal sequences are responsible for vacuolar targetingof gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).

[0194] By the fusion of the appropriate targeting sequences describedabove to transgene sequences of interest it is possible to direct thetransgene product to any organelle or cell compartment. For chloroplasttargeting, for example, the chloroplast signal sequence from the RUBISCOgene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused inframe to the amino terminal ATG of the transgene. The signal sequenceselected should include the known cleavage site, and the fusionconstructed should take into account any amino acids after the cleavagesite which are required for cleavage. In some cases this requirement maybe fulfilled by the addition of a small number of amino acids betweenthe cleavage site and the transgene ATG or, alternatively, replacementof some amino acids within the transgene sequence. Fusions constructedfor chloroplast import can be tested for efficacy of chloroplast uptakeby in vitro translation of in vitro transcribed constructions followedby in vitro chloroplast uptake using techniques described by Bartlett etal. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology,Elsevier pp 1081-1091 (1982) and Wasmann et al. Mol. Gen. Genet. 205:446-453 (1986). These construction techniques are well known in the artand are equally applicable to mitochondria and peroxisomes.

[0195] The above-described mechanisms for cellular targeting can beutilized not only in conjunction with their cognate promoters, but alsoin conjunction with heterologous promoters so as to effect a specificcell-targeting goal under the transcriptional regulation of a promoterthat has an expression pattern different to that of the promoter fromwhich the targeting signal derives.

Example 15 Construction of Plant Transformation Vectors

[0196] Numerous transformation vectors available for planttransformation are known to those of ordinary skill in the planttransformation arts, and the genes pertinent to this invention can beused in conjunction with any such vectors. The selection of vector willdepend upon the preferred transformation technique and the targetspecies for transformation. For certain target species, differentantibiotic or herbicide selection markers may be preferred. Selectionmarkers used routinely in transformation include the nptII gene, whichconfers resistance to kanamycin and related antibiotics (Messing &Vierra. Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187(1983)), the bar gene, which confers resistance to the herbicidephosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spenceret al. Theor. Appl. Genet 79: 625-631 (1990)), the hph gene, whichconfers resistance to the antibiotic hygromycin (Blochinger &Diggelmann, Mol Cell Biol 4: 2929-2931), and the dhfr gene, whichconfers resistance to methatrexate (Bourouis et al., EMBO J. 2(7):1099-1104 (1983)), the EPSPS gene, which confers resistance toglyphosate (U.S. Pat. Nos. 4,940,935 and 5,188,642), and themannose-6-phosphate isomerase gene, which provides the ability tometabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629).

[0197] 1. Vectors Suitable for Agrobacterium Transformation

[0198] Many vectors are available for transformation using Agrobacteriumtumefaciens. These typically carry at least one T-DNA border sequenceand include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) andpXYZ. Below, the construction of two typical vectors suitable forAgrobacterium transformation is described.

[0199] a. pCIB200 and pCIB2001:

[0200] The binary vectors pcIB200 and pCIB2001 are used for theconstruction of recombinant vectors for use with Agrobacterium and areconstructed in the following manner. pTJS75kan is created by NarIdigestion of pTJS75 (Schmidhauser & Helinski, J. Bacteriol. 164: 446-455(1985)) allowing excision of the tetracycline-resistance gene, followedby insertion of an AccI fragment from pUC4K carrying an NPTII (Messing &Vierra, Gene 19: 259-268 (1982): Bevan et al., Nature 304: 184-187(1983): McBride et al., Plant Molecular Biology 14: 266-276 (1990)).XhoI linkers are ligated to the EcoRV fragment of PCIB7 which containsthe left and right T-DNA borders, a plant selectable nos/nptII chimericgene and the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)),and the XhoI-digested fragment are cloned into SaII-digested pTJS75kanto create pCIB200 (see also EP 0 332 104, example 19). pCIB200 containsthe following unique polylinker restriction sites: EcoRI, SstI, KpnI,BgIII, XbaI, and SaII. pCIB2001 is a derivative of pCIB200 created bythe insertion into the polylinker of additional restriction sites.Unique restriction sites in the polylinker of pCIB2001 are EcoRI, SstI,KpnI, BgIII, XbaI, SaII, MIuI, BcII, AvrII, ApaI, HpaI, and StuI.pCIB2001, in addition to containing these unique restriction sites alsohas plant and bacterial kanamycin selection, left and right T-DNAborders for Agrobacterium-mediated transformation, the RK2-derived trfAfunction for mobilization between E. coli and other hosts, and the OriTand OriV functions also from RK2. The pCIB2001 polylinker is suitablefor the cloning of plant expression cassettes containing their ownregulatory signals.

[0201] b. pCIB10 and Hygromycin Selection Derivatives Thereof:

[0202] The binary vector pCIB10 contains a gene encoding kanamycinresistance for selection in plants and T-DNA right and left bordersequences and incorporates sequences from the wide host-range plasmidpRK252 allowing it to replicate in both E. coli and Agrobacterium. Itsconstruction is described by Rothstein et al. (Gene 53: 153-161 (1987)).Various derivatives of pCIB10 are constructed which incorporate the genefor hygromycin B phosphotransferase described by Gritz et al. (Gene 25:179-188 (1983)). These derivatives enable selection of transgenic plantcells on hygromycin only (pCIB743), or hygromycin and kanamycin(pCIB715, pCIB717).

[0203] 2. Vectors Suitable for non-Agrobacterium Transformation

[0204] Transformation without the use of Agrobacterium tumefacienscircumvents the requirement for T-DNA sequences in the chosentransformation vector and consequently vectors lacking these sequencescan be utilized in addition to vectors such as the ones described abovewhich contain T-DNA sequences. Transformation techniques that do notrely on Agrobacterium include transformation via particle bombardment,protoplast uptake (e.g. PEG and electroporation) and microinjection. Thechoice of vector depends largely on the preferred selection for thespecies being transformed. Below, the construction of typical vectorssuitable for non-Agrobacterium transformation is described.

[0205] a. pCIB3064:

[0206] pCIB3064 is a pUC-derived vector suitable for direct genetransfer techniques in combination with selection by the herbicide basta(or phosphinothricin). The plasmid pCIB246 comprises the CaMV 35Spromoter in operational fusion to the E. coli GUS gene and the CaMV 35Stranscriptional terminator and is described in the PCT publishedapplication WO 93/07278. The 35S promoter of this vector contains twoATG sequences 5′ of the start site. These sites are mutated usingstandard PCR techniques in such a way as to remove the ATGs and generatethe restriction sites SspI and PvuII. The new restriction sites are 96and 37 bp away from the unique SaII site and 101 and 42 bp away from theactual start site. The resultant derivative of pCIB246 is designatedpCIB3025. The GUS gene is then excised from pCIB3025 by digestion withSaII and SacI, the termini rendered blunt and religated to generateplasmid pCIB3060. The plasmid pJIT82 is obtained from the John InnesCentre, Norwich and the a 400 bp SmaI fragment containing the bar genefrom Streptomyces viridochromogenes is excised and inserted into theHpaI site of pCIB3060 (Thompson et al. EMBO J 6: 2519-2523 (1987)). Thisgenerated pCIB3064, which comprises the bar gene under the control ofthe CaMV 35S promoter and terminator for herbicide selection, a gene forampicillin resistance (for selection in E. coli) and a polylinker withthe unique sites SphI, PstI, HindIII, and BamHI. This vector is suitablefor the cloning of plant expression cassettes containing their ownregulatory signals.

[0207] b. pSOG19 and pSOG35:

[0208] pSOG35 is a transformation vector that utilizes the E. Coli genedihydrofolate reductase (DFR) as a selectable marker conferringresistance to methotrexate. PCR is used to amplify the 35S promoter(−800 bp), intron 6 from the maize Adh1 gene (−550 bp) and 18 bp of theGUS untranslated leader sequence from pSOG10. A 250-bp fragment encodingthe E. coli dihydrofolate reductase type II gene is also amplified byPCR and these two PCR fragments are assembled with a SacI-PstI fragmentfrom pB1221 (Clontech) which comprises the pUC19 vector backbone and thenopaline synthase terminator. Assembly of these fragments generatespSOG19 which contains the 35S promoter in fusion with the intron 6sequence, the GUS leader, the DHFR gene and the nopaline synthaseterminator. Replacement of the GUS leader in pSOG19 with the leadersequence from Maize Chlorotic Mottle Virus (MCMV) generates the vectorpSOG35. pSOG19 and pSOG35 carry the pUC gene for ampicillin resistanceand have HindIII, SphI, PstI and EcoRI sites available for the cloningof foreign substances.

[0209] 3. Vector Suitable for Chloroplast Transformation

[0210] For expression of a nucleotide sequence of the present inventionin plant plastids, plastid transformation vector pPH143 (WO 97/32011,example 36) is used. The nucleotide sequence is inserted into pPH143thereby replacing the PROTOX coding sequence. This vector is then usedfor plastid transformation and selection of transformants forspectinomycin resistance. Alternatively, the nucleotide sequence isinserted in pPH143 so that it replaces the aadH gene. In this case,transformants are selected for resistance to PROTOX inhibitors.

Example 16 Transformation

[0211] Once a nucleic acid sequence of the invention has been clonedinto an expression system, it is transformed into a plant cell. Methodsfor transformation and regeneration of plants are well known in the art.For example, Ti plasmid vectors have been utilized for the delivery offoreign DNA, as well as direct DNA uptake, liposomes, electroporation,micro-injection, and microprojectiles. In addition, bacteria from thegenus Agrobacterium can be utilized to transform plant cells. Below aredescriptions of representative techniques for transforming bothdicotyledonous and monocotyledonous plants, as well as a representativeplastid transformation technique.

[0212] 1. Transformation of Dicotyledons

[0213] Transformation techniques for dicotyledons are well known in theart and include Agrobacterium-based techniques and techniques that donot require Agrobacterium. Non-Agrobacterium techniques involve theuptake of exogenous genetic material directly by protoplasts or cells.This can be accomplished by PEG or electroporation mediated uptake,particle bombardment-mediated delivery, or microinjection. Examples ofthese techniques are described by Paszkowski et al., EMBO J 3: 2717-2722(1984), Potrykus et al., Mol. Gen. Genet. 199: 169-177 (1985), Reich etal., Biotechnology 4: 1001-1004 (1986), and Klein et al., Nature 327:70-73 (1987). In each case the transformed cells are regenerated towhole plants using standard techniques known in the art.

[0214] Agrobacterium-mediated transformation is a preferred techniquefor transformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species.Agrobacterium transformation typically involves the transfer of thebinary vector carrying the foreign DNA of interest (e.g. pCIB200 orpCIB2001) to an appropriate Agrobacterium strain which may depend of thecomplement of vir genes carried by the host Agrobacterium strain eitheron a co-resident Ti plasmid or chromosomally (e.g. strain CIB542 forpCIB200 and pCIB2001 (Uknes et al. Plant Cell 5: 159-169 (1993)). Thetransfer of the recombinant binary vector to Agrobacterium isaccomplished by a triparental mating procedure using E. coli carryingthe recombinant binary vector, a helper E. coli strain which carries aplasmid such as pRK2013 and which is able to mobilize the recombinantbinary vector to the target Agrobacterium strain. Alternatively, therecombinant binary vector can be transferred to Agrobacterium by DNAtransformation (Höfgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

[0215] Transformation of the target plant species by recombinantAgrobacterium usually involves co-cultivation of the Agrobacterium withexplants from the plant and follows protocols well known in the art.Transformed tissue is regenerated on selectable medium carrying theantibiotic or herbicide resistance marker present between the binaryplasmid T-DNA borders.

[0216] Another approach to transforming plant cells with a gene involvespropelling inert or biologically active particles at plant tissues andcells. This technique is disclosed in U.S. Pat. Nos. 4,945,050,5,036,006, and 5,100,792 all to Sanford et al. Generally, this procedureinvolves propelling inert or biologically active particles at the cellsunder conditions effective to penetrate the outer surface of the celland afford incorporation within the interior thereof. When inertparticles are utilized, the vector can be introduced into the cell bycoating the particles with the vector containing the desired gene.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried yeast cells, dried bacteriumor a bacteriophage, each containing DNA sought to be introduced) canalso be propelled into plant cell tissue.

[0217] 2. Transformation of Monocotyledons

[0218] Transformation of most monocotyledon species has now also becomeroutine. Preferred techniques include direct gene transfer intoprotoplasts using PEG or electroporation techniques, and particlebombardment into callus tissue. Transformations can be undertaken with asingle DNA species or multiple DNA species (i.e. co-transformation) andboth these techniques are suitable for use with this invention.Co-transformation may have the advantage of avoiding complete vectorconstruction and of generating transgenic plants with unlinked loci forthe gene of interest and the selectable marker, enabling the removal ofthe selectable marker in subsequent generations, should this be regardeddesirable. However, a disadvantage of the use of co-transformation isthe less than 100% frequency with which separate DNA species areintegrated into the genome (Schocher et al. Biotechnology 4: 1093-1096(1986)).

[0219] Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278describe techniques for the preparation of callus and protoplasts froman elite inbred line of maize, transformation of protoplasts using PEGor electroporation, and the regeneration of maize plants fromtransformed protoplasts. Gordon-Kamm et al. (Plant Cell 2: 603-618(1990)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) havepublished techniques for transformation of A188-derived maize line usingparticle bombardment. Furthermore, WO 93/07278 and Koziel et al.(Biotechnology 11: 194-200 (1993)) describe techniques for thetransformation of elite inbred lines of maize by particle bombardment.This technique utilizes immature maize embryos of 1.5-2.5 mm lengthexcised from a maize ear 14-15 days after pollination and a PDS-1000HeBiolistics device for bombardment.

[0220] Transformation of rice can also be undertaken by direct genetransfer techniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhang et al. Plant Cell Rep 7: 379-384 (1988);Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology8: 736-740 (1990)). Both types are also routinely transformable usingparticle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)).Furthermore, WO 93/21335 describes techniques for the transformation ofrice via electroporation.

[0221] Patent Application EP 0 332 581 describes techniques for thegeneration, transformation and regeneration of Pooideae protoplasts.These techniques allow the transformation of Dactylis and wheat.Furthermore, wheat transformation has been described by Vasil et al.(Biotechnology 10: 667-674 (1992)) using particle bombardment into cellsof type C long-term regenerable callus, and also by Vasil et al.(Biotechnology 11: 1553-1558 (1993)) and Weeks et al. (Plant Physiol.102: 1077-1084 (1993)) using particle bombardment of immature embryosand immature embryo-derived callus. A preferred technique for wheattransformation, however, involves the transformation of wheat byparticle bombardment of immature embryos and includes either a highsucrose or a high maltose step prior to gene delivery. Prior tobombardment, any number of embryos (0.75-1 mm in length) are plated ontoMS medium with 3% sucrose (Murashiga & Skoog, Physiologia Plantarum 15:473-497 (1962)) and 3 mg/l 2,4-D for induction of somatic embryos, whichis allowed to proceed in the dark. On the chosen day of bombardment,embryos are removed from the induction medium and placed onto theosmoticum (i.e. induction medium with sucrose or maltose added at thedesired concentration, typically 15%). The embryos are allowed toplasmolyze for 2-3 h and are then bombarded. Twenty embryos per targetplate is typical, although not critical. An appropriate gene-carryingplasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer sizegold particles using standard procedures. Each plate of embryos is shotwith the DuPont Biolistics® helium device using a burst pressure of˜1000 psi using a standard 80 mesh screen. After bombardment, theembryos are placed back into the dark to recover for about 24 h (stillon osmoticum). After 24 hrs, the embryos are removed from the osmoticumand placed back onto induction medium where they stay for about a monthbefore regeneration. Approximately one month later the embryo explantswith developing embryogenic callus are transferred to regenerationmedium (MS+1 mg/liter NM, 5 mg/liter GA), further containing theappropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2mg/l methotrexate in the case of pSOG35). After approximately one month,developed shoots are transferred to larger sterile containers known as“GA7s” which contain half-strength MS, 2% sucrose, and the sameconcentration of selection agent.

[0222] Transformation of monocotyledons using Agrobacterium has alsobeen described. See, WO 94/00977 and U.S. Pat. No. 5,591,616, which areincorporated herein by reference.

[0223] 3. Transformation of Plastids

[0224] Seeds of Nicotiana tabacum c.v. ‘Xanthi nc’ are germinated sevenper plate in a 1″ circular array on T agar medium and bombarded 12-14days after sowing with 1 μm tungsten particles (M10, Biorad, Hercules,Calif.) coated with DNA from plasmids pPH143 and pPH145 essentially asdescribed (Svab, Z. and Maliga, P. (1993) PNAS 90, 913-917). Bombardedseedlings are incubated on T medium for two days after which leaves areexcised and placed abaxial side up in bright light (350-500 μmolphotons/m²/s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. andMaliga, P. (1990) PNAS 87, 8526-8530) containing 500 μg/ml spectinomycindihydrochloride (Sigma, St. Louis, Mo.). Resistant shoots appearingunderneath the bleached leaves three to eight weeks after bombardmentare subcloned onto the same selective medium, allowed to form callus,and secondary shoots isolated and subcloned. Complete segregation oftransformed plastid genome copies (homoplasmicity) in independentsubclones is assessed by standard techniques of Southern blotting(Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor). BamHI/EcoRI-digestedtotal cellular DNA (Mettler, I. J. (1987) Plant Mol Biol Reporter 5,346-349) is separated on 1% Tris-borate (TBE) agarose gels, transferredto nylon membranes (Amersham) and probed with ³²P-labeled random primedDNA sequences corresponding to a 0.7 kb BamHI/HindIII DNA fragment frompC8 containing a portion of the rps7/12 plastid targeting sequence.Homoplasmic shoots are rooted aseptically on spectinomycin-containingMS/IBA medium (McBride, K. E. et al. (1994) PNAS 91, 7301-7305) andtransferred to the greenhouse.

E. Breeding and Seed Production Example 17 Breeding

[0225] The plants obtained via transformation with a nucleic acidsequence of the present invention can be any of a wide variety of plantspecies, including those of monocots and dicots; however, the plantsused in the method of the invention are preferably selected from thelist of agronomically important target crops set forth supra. Theexpression of a gene of the present invention in combination with othercharacteristics important for production and quality can be incorporatedinto plant lines through breeding. Breeding approaches and techniquesare known in the art. See, for example, Welsh J. R., Fundamentals ofPlant Genetics and Breeding, John Wiley & Sons, NY (1981); CropBreeding, Wood D. R. (Ed.) American Society of Agronomy Madison, Wis.(1983); Mayo O., The Theory of Plant Breeding, Second Edition, ClarendonPress, Oxford (1987); Singh, D. P., Breeding for Resistance to Diseasesand Insect Pests, Springer-Verlag, NY (1986); and Wricke and Weber,Quantitative Genetics and Selection Plant Breeding, Walter de Gruyterand Co., Berlin (1986).

[0226] The genetic properties engineered into the transgenic seeds andplants described above are passed on by sexual reproduction orvegetative growth and can thus be maintained and propagated in progenyplants. Generally said maintenance and propagation make use of knownagricultural methods developed to fit specific purposes such as tilling,sowing or harvesting. Specialized processes such as hydroponics orgreenhouse technologies can also be applied. As the growing crop isvulnerable to attack and damages caused by insects or infections as wellas to competition by weed plants, measures are undertaken to controlweeds, plant diseases, insects, nematodes, and other adverse conditionsto improve yield. These include mechanical measures such a tillage ofthe soil or removal of weeds and infected plants, as well as theapplication of agrochemicals such as herbicides, fungicides,gametocides, nematicides, growth regulants, ripening agents andinsecticides.

[0227] Use of the advantageous genetic properties of the transgenicplants and seeds according to the invention can further be made in plantbreeding, which aims at the development of plants with improvedproperties such as tolerance of pests, herbicides, or stress, improvednutritional value, increased yield, or improved structure causing lessloss from lodging or shattering. The various breeding steps arecharacterized by well-defined human intervention such as selecting thelines to be crossed, directing pollination of the parental lines, orselecting appropriate progeny plants. Depending on the desiredproperties, different breeding measures are taken. The relevanttechniques are well known in the art and include but are not limited tohybridization, inbreeding, backcross breeding, multiline breeding,variety blend, interspecific hybridization, aneuploid techniques, etc.Hybridization techniques also include the sterilization of plants toyield male or female sterile plants by mechanical, chemical, orbiochemical means. Cross pollination of a male sterile plant with pollenof a different line assures that the genome of the male sterile butfemale fertile plant will uniformly obtain properties of both parentallines. Thus, the transgenic seeds and plants according to the inventioncan be used for the breeding of improved plant lines, that for example,increase the effectiveness of conventional methods such as herbicide orpesticide treatment or allow one to dispense with said methods due totheir modified genetic properties. Alternatively new crops with improvedstress tolerance can be obtained, which, due to their optimized genetic“equipment”, yield harvested product of better quality than productsthat were not able to tolerate comparable adverse developmentalconditions.

Example 18 Seed Production

[0228] In seed production, germination quality and uniformity of seedsare essential product characteristics, whereas germination quality anduniformity of seeds harvested and sold by the farmer is not important.As it is difficult to keep a crop free from other crop and weed seeds,to control seedborne diseases, and to produce seed with goodgermination, fairly extensive and well-defined seed production practiceshave been developed by seed producers, who are experienced in the art ofgrowing, conditioning and marketing of pure seed. Thus, it is commonpractice for the farmer to buy certified seed meeting specific qualitystandards instead of using seed harvested from his own crop. Propagationmaterial to be used as seeds is customarily treated with a protectantcoating comprising herbicides, insecticides, fungicides, bactericides,nematicides, molluscicides, or mixtures thereof. Customarily usedprotectant coatings comprise compounds such as captan, carboxin, thiram(TMTD®), methalaxyl (Apron®), and pirimiphos-methyl (Actellic®). Ifdesired, these compounds are formulated together with further carriers,surfactants or application-promoting adjuvants customarily employed inthe art of formulation to provide protection against damage caused bybacterial, fungal or animal pests. The protectant coatings may beapplied by impregnating propagation material with a liquid formulationor by coating with a combined wet or dry formulation. Other methods ofapplication are also possible such as treatment directed at the buds orthe fruit.

[0229] It is a further aspect of the present invention to provide newagricultural methods, such as the methods exemplified above, which arecharacterized by the use of transgenic plants, transgenic plantmaterial, or transgenic seed according to the present invention.

[0230] The seeds may be provided in a bag, container or vessel comprisedof a suitable packaging material, the bag or container capable of beingclosed to contain seeds. The bag, container or vessel may be designedfor either short term or long term storage, or both, of the seed.Examples of a suitable packaging material include paper, such as kraftpaper, rigid or pliable plastic or other polymeric material, glass ormetal. Desirably the bag, container, or vessel is comprised of aplurality of layers of packaging materials, of the same or differingtype. In one embodiment the bag, container or vessel is provided so asto exclude or limit water and moisture from contacting the seed. In oneexample, the bag, container or vessel is sealed, for example heatsealed, to prevent water or moisture from entering. In anotherembodiment water absorbent materials are placed between or adjacent topackaging material layers. In yet another embodiment the bag, containeror vessel, or packaging material of which it is comprised is treated tolimit, suppress or prevent disease, contamination or other adverseaffects of storage or transport of the seed. An example of suchtreatment is sterilization, for example by chemical means or by exposureto radiation. Comprised by the present invention is a commercial bagcomprising seed of a transgenic plant comprising a gene of the presentinvention that is expressed in said transformed plant at higher levelsthan in a wild type plant, together with a suitable carrier, togetherwith label instructions for the use thereof for conferring broadspectrum disease resistance to plants.

[0231] The above disclosed embodiments are illustrative. This disclosureof the invention will place one skilled in the art in possession of manyvariations of the invention. All such obvious and foreseeable variationsare intended to be encompassed by the appended claims.

1 5 1 6930 DNA Bacillus thuringiensis source (1)..(6930) Bacillusthuringiensis subsp. finitimus strain VKPMG-1166 (taxon29337 1gatccagccg cttctgtttt tggtaacgat gattgtttcc agtcttatgg tgaattgggg 60ataggtaatt ttacaagccc tgaaacattt atagatgcac aaggggctat tagtacagca 120atcagtgtaa ctggaacaat actcggattt ttaggggttc catttgctgg tcaaatcaca 180gctttttacc agaaggtatt aggattattg tggccaaatc aacaaacgaa acaatgggaa 240gagtttatga aacaagttga ggctctcatc gataaaaaaa tatctgaggc tgtgcggagt 300aaagctattg cggaattgca agggttaggt aataatttag acctatatac ggaggctctt 360gaagaatggc tagaaaacaa ggagagcccg tgaaaacgtg accgtgtgat tcagagatgg 420cgtaatgcag atagtctttt tgaacaattt atgccctctt ttcaatcaaa tgggtttgaa 480gtactgttat taacagtcta tgctcaagca gcaaatttac atttgctttt attaagagat 540tgttctattt atggagctga atggggatta accccatgcc catcaactta agggggaaat 600aaacgaacct tcattcagcc aggttaaaac attcttaaaa tcttctactt taataattat 660taattcttaa taacctacat tctagctcta tatttacctt taacagtgca atttttttgt 720ttggaggata cttcaaaatg ttaagttgat ggatgtgtgg ttctacccct agtatgtgca 780cagaataata atgtaaagca taagataaag aatcattcaa tttgtattaa taaataccgt 840ttgttgtagg aagatgtatt ctttttttat tatctattaa aattggagga atttga atg 899Met 1 aat agc gaa gaa atg aat cat gta aac cca ttt gaa ata tca gat aat947 Asn Ser Glu Glu Met Asn His Val Asn Pro Phe Glu Ile Ser Asp Asn 5 1015 aat gat gtc tcc ata cct tct caa aga tat cca ttt gca aat gat cca 995Asn Asp Val Ser Ile Pro Ser Gln Arg Tyr Pro Phe Ala Asn Asp Pro 20 25 30gca gat tcg gtt ttt tgt gca gat gat ttt tta cag tct tat ggt gaa 1043 AlaAsp Ser Val Phe Cys Ala Asp Asp Phe Leu Gln Ser Tyr Gly Glu 35 40 45 tttaat atg gat aat ttc ggg gaa tcc gaa cct ttt ata gat gca tca 1091 Phe AsnMet Asp Asn Phe Gly Glu Ser Glu Pro Phe Ile Asp Ala Ser 50 55 60 65 ggcgcc att aat gcg gca att ggt gta act gga aca gta ctc gga ttc 1139 Gly AlaIle Asn Ala Ala Ile Gly Val Thr Gly Thr Val Leu Gly Phe 70 75 80 tta ggtgtt cca ttt gca ggt gct ctt aca aca ttt tat caa aaa tta 1187 Leu Gly ValPro Phe Ala Gly Ala Leu Thr Thr Phe Tyr Gln Lys Leu 85 90 95 ttt ggt tttttg ttt cca aat aac aat act aaa caa tgg gaa gaa ttt 1235 Phe Gly Phe LeuPhe Pro Asn Asn Asn Thr Lys Gln Trp Glu Glu Phe 100 105 110 atg aaa caagtt gag gca ctc atc gat gaa aaa ata tct gat gct gtg 1283 Met Lys Gln ValGlu Ala Leu Ile Asp Glu Lys Ile Ser Asp Ala Val 115 120 125 cga aat aaggct att tca gaa tta caa ggg tta gtt aat aat ata act 1331 Arg Asn Lys AlaIle Ser Glu Leu Gln Gly Leu Val Asn Asn Ile Thr 130 135 140 145 cta tataca gag gcc ctt gaa gaa tgg tta gaa aat aag gaa aat cct 1379 Leu Tyr ThrGlu Ala Leu Glu Glu Trp Leu Glu Asn Lys Glu Asn Pro 150 155 160 gca gtacgt gat cgt gtt ctt cag cga tgg cgg att ctg gat ggt ttt 1427 Ala Val ArgAsp Arg Val Leu Gln Arg Trp Arg Ile Leu Asp Gly Phe 165 170 175 ttt gaacaa cag atg cct tct ttt gca gta aag gga ttt gaa gta ctt 1475 Phe Glu GlnGln Met Pro Ser Phe Ala Val Lys Gly Phe Glu Val Leu 180 185 190 tta ttggta gta tat act cag gcc gca aat tta cat tta ctt tca cta 1523 Leu Leu ValVal Tyr Thr Gln Ala Ala Asn Leu His Leu Leu Ser Leu 195 200 205 aga gatgct tat ata tac ggg gcg gag tgg gga tta act cca aca aac 1571 Arg Asp AlaTyr Ile Tyr Gly Ala Glu Trp Gly Leu Thr Pro Thr Asn 210 215 220 225 attgat caa aac cac aca aga ttg tta cgt cat tcc gca gag tac act 1619 Ile AspGln Asn His Thr Arg Leu Leu Arg His Ser Ala Glu Tyr Thr 230 235 240 gatcac tgt gta aat tgg tat aat acc ggc tta aaa caa tta gag aat 1667 Asp HisCys Val Asn Trp Tyr Asn Thr Gly Leu Lys Gln Leu Glu Asn 245 250 255 tccgat gca aaa agc tgg ttc caa tat aat cgt ttc cgc aga gaa atg 1715 Ser AspAla Lys Ser Trp Phe Gln Tyr Asn Arg Phe Arg Arg Glu Met 260 265 270 actctt tct gta tta gat gtt atc gca ttg ttc cct gcg tat gat gtg 1763 Thr LeuSer Val Leu Asp Val Ile Ala Leu Phe Pro Ala Tyr Asp Val 275 280 285 aaaatg tat cca ata cca aca aat ttt cag ctt act cga gaa gtg tat 1811 Lys MetTyr Pro Ile Pro Thr Asn Phe Gln Leu Thr Arg Glu Val Tyr 290 295 300 305aca gat gta ata ggt aaa att gga aga aat gat agc gac cat tgg tat 1859 ThrAsp Val Ile Gly Lys Ile Gly Arg Asn Asp Ser Asp His Trp Tyr 310 315 320agt gcc aat gcc cct tca ttt tca aat ctt gaa agt acc tta ata cga 1907 SerAla Asn Ala Pro Ser Phe Ser Asn Leu Glu Ser Thr Leu Ile Arg 325 330 335aca cct cat gtg gta gat tat ata aaa aaa cta aaa att ttt tat gcc 1955 ThrPro His Val Val Asp Tyr Ile Lys Lys Leu Lys Ile Phe Tyr Ala 340 345 350act gtt gat tat tat gga atc tat gga cga tct ggg aaa tgg gtt ggt 2003 ThrVal Asp Tyr Tyr Gly Ile Tyr Gly Arg Ser Gly Lys Trp Val Gly 355 360 365cat ata ata aca tct gca act tct gcg aat acg aca gaa acc cgt aac 2051 HisIle Ile Thr Ser Ala Thr Ser Ala Asn Thr Thr Glu Thr Arg Asn 370 375 380385 tat gga acg ata gta aat cat gat agt gtt gag ttg aac ttt gaa ggg 2099Tyr Gly Thr Ile Val Asn His Asp Ser Val Glu Leu Asn Phe Glu Gly 390 395400 aaa aat att tat aaa acg gga tcg ctg cca cag gga gtt cct cct tac 2147Lys Asn Ile Tyr Lys Thr Gly Ser Leu Pro Gln Gly Val Pro Pro Tyr 405 410415 caa att ggc tat gtt act cct att tat ttt ata act agg gcc gtt aac 2195Gln Ile Gly Tyr Val Thr Pro Ile Tyr Phe Ile Thr Arg Ala Val Asn 420 425430 ttt ttt aca gta tca ggt tcc aaa act tcc gta gag aaa tat tac tca 2243Phe Phe Thr Val Ser Gly Ser Lys Thr Ser Val Glu Lys Tyr Tyr Ser 435 440445 aaa aaa gac aga tat tat agt gaa gga ctg cca gag gag cag ggg gtt 2291Lys Lys Asp Arg Tyr Tyr Ser Glu Gly Leu Pro Glu Glu Gln Gly Val 450 455460 465 ttt tca acc gaa caa ctg cca cct aat agt ata gcg gaa cca gaa cat2339 Phe Ser Thr Glu Gln Leu Pro Pro Asn Ser Ile Ala Glu Pro Glu His 470475 480 ata gcg tac agc cat cgt cta tgt cat gtt act ttc att agt gtt tcc2387 Ile Ala Tyr Ser His Arg Leu Cys His Val Thr Phe Ile Ser Val Ser 485490 495 aat ggc aat aag tat tca aaa gat cta cca tta ttt tca tgg acg cat2435 Asn Gly Asn Lys Tyr Ser Lys Asp Leu Pro Leu Phe Ser Trp Thr His 500505 510 tct agt gta gat ttc gat aat tat gtt tat ccg aca aag att act cag2483 Ser Ser Val Asp Phe Asp Asn Tyr Val Tyr Pro Thr Lys Ile Thr Gln 515520 525 ctt cct gcg aca aaa gga tac aat gtg tcc ata gta aaa gaa cca gga2531 Leu Pro Ala Thr Lys Gly Tyr Asn Val Ser Ile Val Lys Glu Pro Gly 530535 540 545 ttt att ggg gga gat ata ggc aag aat aat ggt caa att tta gggaaa 2579 Phe Ile Gly Gly Asp Ile Gly Lys Asn Asn Gly Gln Ile Leu Gly Lys550 555 560 tac aaa gtt aac gta gaa gat gtt tct caa aaa tat aga ttt agagtc 2627 Tyr Lys Val Asn Val Glu Asp Val Ser Gln Lys Tyr Arg Phe Arg Val565 570 575 cga tat gct act gaa aca gaa ggt gaa tta ggt ata aaa ata gatggc 2675 Arg Tyr Ala Thr Glu Thr Glu Gly Glu Leu Gly Ile Lys Ile Asp Gly580 585 590 cgt acg gtt aat tta tat caa tat aaa aaa acc aaa gca ccc ggagat 2723 Arg Thr Val Asn Leu Tyr Gln Tyr Lys Lys Thr Lys Ala Pro Gly Asp595 600 605 cct tta aca tac aaa gcg ttt gat tat ttg tct ttt tca acc ccagtt 2771 Pro Leu Thr Tyr Lys Ala Phe Asp Tyr Leu Ser Phe Ser Thr Pro Val610 615 620 625 aaa ttt aac aat gcc tca tca aca att gaa tta ttt tta caaaat aaa 2819 Lys Phe Asn Asn Ala Ser Ser Thr Ile Glu Leu Phe Leu Gln AsnLys 630 635 640 acc tca gga act ttt tat cta gct gga ata gag ata ata ccagta aaa 2867 Thr Ser Gly Thr Phe Tyr Leu Ala Gly Ile Glu Ile Ile Pro ValLys 645 650 655 agt aat tat gag gag gag ctt act ctt gaa gaa gcg aaa aaggca gtg 2915 Ser Asn Tyr Glu Glu Glu Leu Thr Leu Glu Glu Ala Lys Lys AlaVal 660 665 670 agt agt ttg ttc aca gat gca aga aat gca ttg aaa ata gatgtg aca 2963 Ser Ser Leu Phe Thr Asp Ala Arg Asn Ala Leu Lys Ile Asp ValThr 675 680 685 gat tac caa att gat caa gcg gca aat tta gta gaa tgt atatcg ggt 3011 Asp Tyr Gln Ile Asp Gln Ala Ala Asn Leu Val Glu Cys Ile SerGly 690 695 700 705 gac ctg tat gca aaa gag aaa ata gtg tta ctt cgt gctgtt aag ttt 3059 Asp Leu Tyr Ala Lys Glu Lys Ile Val Leu Leu Arg Ala ValLys Phe 710 715 720 gcg aaa caa ttg agt caa tcc caa aat tta tta tca gaccct gaa ttt 3107 Ala Lys Gln Leu Ser Gln Ser Gln Asn Leu Leu Ser Asp ProGlu Phe 725 730 735 aac aat gtg aat aga gaa aat agc tgg aca gca agt acaagt gtc gca 3155 Asn Asn Val Asn Arg Glu Asn Ser Trp Thr Ala Ser Thr SerVal Ala 740 745 750 atc att gaa gga gac cca ttg tat aaa ggg cgc gct gttcaa tta tca 3203 Ile Ile Glu Gly Asp Pro Leu Tyr Lys Gly Arg Ala Val GlnLeu Ser 755 760 765 agt gcg agg gat gaa aac ttt cca aca tat tta tac cagaag ata gat 3251 Ser Ala Arg Asp Glu Asn Phe Pro Thr Tyr Leu Tyr Gln LysIle Asp 770 775 780 785 gaa tcc aca tta aaa cca tat aca cgt tat caa ctaaga gga ttt gta 3299 Glu Ser Thr Leu Lys Pro Tyr Thr Arg Tyr Gln Leu ArgGly Phe Val 790 795 800 gaa ggc agt gaa aat tta gat gtc tac ttg atc cgttat ggc gca gca 3347 Glu Gly Ser Glu Asn Leu Asp Val Tyr Leu Ile Arg TyrGly Ala Ala 805 810 815 cat gta aga atg aat gtg cct tat aat ctt gaa ataatc gat act tct 3395 His Val Arg Met Asn Val Pro Tyr Asn Leu Glu Ile IleAsp Thr Ser 820 825 830 tca cct gta aat cct tgt gaa gag gta gac ggt ctatct cat cgt tcg 3443 Ser Pro Val Asn Pro Cys Glu Glu Val Asp Gly Leu SerHis Arg Ser 835 840 845 tgc aac gta ttt gat cgc tgt aag cag tct att tctgta gcc ccg gac 3491 Cys Asn Val Phe Asp Arg Cys Lys Gln Ser Ile Ser ValAla Pro Asp 850 855 860 865 gca aat aca gga cct gat cag atc gat gga gatcca cac gcc ttt tct 3539 Ala Asn Thr Gly Pro Asp Gln Ile Asp Gly Asp ProHis Ala Phe Ser 870 875 880 ttc cat att gat aca gga act gta gat agt actgaa aat cta ggg att 3587 Phe His Ile Asp Thr Gly Thr Val Asp Ser Thr GluAsn Leu Gly Ile 885 890 895 tgg gtt gcc ttt aaa att tct gaa cta gat ggttct gca ata ttt ggt 3635 Trp Val Ala Phe Lys Ile Ser Glu Leu Asp Gly SerAla Ile Phe Gly 900 905 910 aac ctt gaa ttg ata gaa gtg ggt cca tta tctggc gaa gcg tta gca 3683 Asn Leu Glu Leu Ile Glu Val Gly Pro Leu Ser GlyGlu Ala Leu Ala 915 920 925 cag gta caa aga aaa gaa gaa aag tgg aaa caagta ctt gcg aaa aaa 3731 Gln Val Gln Arg Lys Glu Glu Lys Trp Lys Gln ValLeu Ala Lys Lys 930 935 940 945 cgt gaa acg act gcg caa act gta tgc agcggc gaa gca agc caa ttg 3779 Arg Glu Thr Thr Ala Gln Thr Val Cys Ser GlyGlu Ala Ser Gln Leu 950 955 960 acc aac tct tcg cag att ctc aaa ata cgaaat tac gat ttg ata cag 3827 Thr Asn Ser Ser Gln Ile Leu Lys Ile Arg AsnTyr Asp Leu Ile Gln 965 970 975 aat ttt cga ata ttc tct ctg cgg aac accttg tct ata aaa ttc aag 3875 Asn Phe Arg Ile Phe Ser Leu Arg Asn Thr LeuSer Ile Lys Phe Lys 980 985 990 ata tat aca ata acg aac tat ccg tat tccagg ctc aat tat gac ttg 3923 Ile Tyr Thr Ile Thr Asn Tyr Pro Tyr Ser ArgLeu Asn Tyr Asp Leu 995 1000 1005 ttt atg gaa cta gag aat aga atc caaaac gca tca ctt tat atg 3968 Phe Met Glu Leu Glu Asn Arg Ile Gln Asn AlaSer Leu Tyr Met 1010 1015 1020 acg tcg aat att ctg caa aat gga gga tttaaa agt gat gta aca 4013 Thr Ser Asn Ile Leu Gln Asn Gly Gly Phe Lys SerAsp Val Thr 1025 1030 1035 agc tgg gaa aca aca gca aat gca gag gta cagcaa ata gac ggt 4058 Ser Trp Glu Thr Thr Ala Asn Ala Glu Val Gln Gln IleAsp Gly 1040 1045 1050 gca tcc gtt tta gtc cta tcg aat tgg aat gca tctgtt gct caa 4103 Ala Ser Val Leu Val Leu Ser Asn Trp Asn Ala Ser Val AlaGln 1055 1060 1065 tct gtt aat gta cag aat gat cat ggc tat gta tta cgtgtc aca 4148 Ser Val Asn Val Gln Asn Asp His Gly Tyr Val Leu Arg Val Thr1070 1075 1080 gca aaa aaa gag ggc att gga aat ggg tat gtc aca atc ttagac 4193 Ala Lys Lys Glu Gly Ile Gly Asn Gly Tyr Val Thr Ile Leu Asp1085 1090 1095 tgt gcc aat cac att gat acc ctg acg ttt agt gct tgt cgctca 4238 Cys Ala Asn His Ile Asp Thr Leu Thr Phe Ser Ala Cys Arg Ser1100 1105 1110 gat tct gat act tcc tct aat gag ctt aca gct tat gta acgaaa 4283 Asp Ser Asp Thr Ser Ser Asn Glu Leu Thr Ala Tyr Val Thr Lys1115 1120 1125 aca cta gaa att ttc ccg gat aca gaa caa att cgt att gaaatc 4328 Thr Leu Glu Ile Phe Pro Asp Thr Glu Gln Ile Arg Ile Glu Ile1130 1135 1140 ggc gaa acg gaa ggt atg ttt tat gta gaa agt gta gag ttaatc 4373 Gly Glu Thr Glu Gly Met Phe Tyr Val Glu Ser Val Glu Leu Ile1145 1150 1155 cga atg gaa aat taa ttagtgaagt tgtaatatcc taaaagcaaaggcggggtct 4428 Arg Met Glu Asn 1160 ctaatgaaga cctcgccttt ttttttaacatgaacgttct aggggttcag ccgagatacg 4488 tgtaaaaacg ggtcatagac tggtttttatttccatgtca tgacccattt atcttttata 4548 caggtatatt ctcttctatc ggttgtggaatgttttgtaa atcaatcaca taatcgccaa 4608 aacgttttat atgtttcgtc atatatgggcttaacatcac cagatcttct cgtgagaatc 4668 gataaccttc tcttttgagt tgccataaaatcatggtaat atcaacgaca ttttgaaaaa 4728 tgactgcatt ggcaacgaga tcattatatttaatacgttt ttcttgctct actggatcat 4788 tttcagtaat aatcccatcc ccaccaaagaataaccactt tgaaaaacca ttatatgctt 4848 ccactttatt tgtactagct gtaatttgctctcttaattt catatctgag atatatctaa 4908 gaagaaatat cgttcgaatg actctgcctagctctcgaaa tgcttggtac agtcgaattc 4968 ttttactatt acttctctag tttctgagaagagttgaggg tagtatcttt ccagctttat 5028 agaaaaacaa cttgaaatta aatcttaccaatgagtctta attggcttgc caacgattac 5088 atcactaaat aaaggtctat atgtatatattgaatatgtt tgcttggtct aaaaaatttt 5148 aagtccttcc aattcctaat tctaggcattaaattgattc ctaatagata agaaagtgca 5208 aagacaggag tagactgacc ttgtgtatcagcatgaatcg tatcgggttg gatatctgac 5268 ttgtttttga gcaatccatc aataatataaacagcttccc aaaccccaca ggggataaaa 5328 tggctaaata gcgcaatata attatccgaaacatgatgat aagctattcc tccatagccc 5388 ccatatctcg atgtggtatt ctgacagtacgttttcttca tataaactcg tattttgttc 5448 catcagccgc ggctgttttt ccgctcccccatagctttgg aaggctaaag aggttgtatt 5508 gatttaaaat gtcttgaatt gaggcatctaatttttgggc actgatatgt ctacgattca 5568 caaaagaaat catatgaggt gttactatatttcgcatatg tttagcggtt tgggctggtc 5628 ctaaattaca gccatatcca aatgaagtaataatataacg ttcaattgga tgctctaatt 5688 taggttcaga tcctgaaaat gggccgaaatgtcttgtaca atgagtccag tgttcaacgt 5748 tacaaagtat atcgagaatc gttcgttccggtagtcgttg tataatttct ttttctaaca 5808 ttttactact cttggaagac tcttttcgtaccaaacgttt caggattggt tctccatctt 5868 cagttatgat aacttgtcca ttattaggatagtttcgatc aacagttttt gcagcggatg 5928 tgagccaact ttttaaccga ttaacaaaatcagtaggtga ggaaggtaat tctagttcat 5988 tacaataatc ttcaaccatc ggttggcattcttcccatgg aagtaattgt tgtctataat 6048 cagcatattt ctcagaacct tttacactaaggtctcccgt ctttaattca taggctaaat 6108 aagaaaatat acagatttca agacgtttacggtaaagtaa attcgtgctt tttcaacgcg 6168 aattgtatgt ttccacagtt cactggcgaatgataaatca atatcgctag gtagatgttc 6228 aaccttacgg ttttcgttct ctaacaaaaattccagagca tttatgaggg aaccgtcttg 6288 agttgttgag tcaatttcta ataatgtgatgagccgaaat aatgtttttc tatgattctt 6348 atagaatttt tctaaaagtg gtagataattatttccgttg tatgaggata ttgcagtaca 6408 gtcttgtttt aaaaattcta taccacctttagcttcaaac agctcagtaa ttttgctgcc 6468 aatttgtgca ttatcatcat gcatagctgttgtttctaaa acttcactca gtacagaaat 6528 aagattttca cttacggaac gatgttgctccctcaaaaga gaaagttctt cttttccttt 6588 tttatgaatg attcccatcc tttttagaaacatttctaca aggttatccc tagttgtaat 6648 ttgtgaccga taaatagttg ataacaacagcatatatctt ttgggaggag cgaagtcctt 6708 tatccctgaa gcatctaacg aattcgcttcagcagcaaag tgtttcaatt tagaatttgg 6768 aatgccttct aataaatcat gaatgtttggaaggaaagac gtaataaatg aaagtctgtt 6828 ttgtaaatct ttcatatgag taatagaagggcttttggga acttctttaa aataattata 6888 acgagaatgt tgaatttctt ttgacgtatgaagtaattga tc 6930 2 1163 PRT Bacillus thuringiensis 2 Met Asn Ser GluGlu Met Asn His Val Asn Pro Phe Glu Ile Ser Asp 1 5 10 15 Asn Asn AspVal Ser Ile Pro Ser Gln Arg Tyr Pro Phe Ala Asn Asp 20 25 30 Pro Ala AspSer Val Phe Cys Ala Asp Asp Phe Leu Gln Ser Tyr Gly 35 40 45 Glu Phe AsnMet Asp Asn Phe Gly Glu Ser Glu Pro Phe Ile Asp Ala 50 55 60 Ser Gly AlaIle Asn Ala Ala Ile Gly Val Thr Gly Thr Val Leu Gly 65 70 75 80 Phe LeuGly Val Pro Phe Ala Gly Ala Leu Thr Thr Phe Tyr Gln Lys 85 90 95 Leu PheGly Phe Leu Phe Pro Asn Asn Asn Thr Lys Gln Trp Glu Glu 100 105 110 PheMet Lys Gln Val Glu Ala Leu Ile Asp Glu Lys Ile Ser Asp Ala 115 120 125Val Arg Asn Lys Ala Ile Ser Glu Leu Gln Gly Leu Val Asn Asn Ile 130 135140 Thr Leu Tyr Thr Glu Ala Leu Glu Glu Trp Leu Glu Asn Lys Glu Asn 145150 155 160 Pro Ala Val Arg Asp Arg Val Leu Gln Arg Trp Arg Ile Leu AspGly 165 170 175 Phe Phe Glu Gln Gln Met Pro Ser Phe Ala Val Lys Gly PheGlu Val 180 185 190 Leu Leu Leu Val Val Tyr Thr Gln Ala Ala Asn Leu HisLeu Leu Ser 195 200 205 Leu Arg Asp Ala Tyr Ile Tyr Gly Ala Glu Trp GlyLeu Thr Pro Thr 210 215 220 Asn Ile Asp Gln Asn His Thr Arg Leu Leu ArgHis Ser Ala Glu Tyr 225 230 235 240 Thr Asp His Cys Val Asn Trp Tyr AsnThr Gly Leu Lys Gln Leu Glu 245 250 255 Asn Ser Asp Ala Lys Ser Trp PheGln Tyr Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Ser Val Leu AspVal Ile Ala Leu Phe Pro Ala Tyr Asp 275 280 285 Val Lys Met Tyr Pro IlePro Thr Asn Phe Gln Leu Thr Arg Glu Val 290 295 300 Tyr Thr Asp Val IleGly Lys Ile Gly Arg Asn Asp Ser Asp His Trp 305 310 315 320 Tyr Ser AlaAsn Ala Pro Ser Phe Ser Asn Leu Glu Ser Thr Leu Ile 325 330 335 Arg ThrPro His Val Val Asp Tyr Ile Lys Lys Leu Lys Ile Phe Tyr 340 345 350 AlaThr Val Asp Tyr Tyr Gly Ile Tyr Gly Arg Ser Gly Lys Trp Val 355 360 365Gly His Ile Ile Thr Ser Ala Thr Ser Ala Asn Thr Thr Glu Thr Arg 370 375380 Asn Tyr Gly Thr Ile Val Asn His Asp Ser Val Glu Leu Asn Phe Glu 385390 395 400 Gly Lys Asn Ile Tyr Lys Thr Gly Ser Leu Pro Gln Gly Val ProPro 405 410 415 Tyr Gln Ile Gly Tyr Val Thr Pro Ile Tyr Phe Ile Thr ArgAla Val 420 425 430 Asn Phe Phe Thr Val Ser Gly Ser Lys Thr Ser Val GluLys Tyr Tyr 435 440 445 Ser Lys Lys Asp Arg Tyr Tyr Ser Glu Gly Leu ProGlu Glu Gln Gly 450 455 460 Val Phe Ser Thr Glu Gln Leu Pro Pro Asn SerIle Ala Glu Pro Glu 465 470 475 480 His Ile Ala Tyr Ser His Arg Leu CysHis Val Thr Phe Ile Ser Val 485 490 495 Ser Asn Gly Asn Lys Tyr Ser LysAsp Leu Pro Leu Phe Ser Trp Thr 500 505 510 His Ser Ser Val Asp Phe AspAsn Tyr Val Tyr Pro Thr Lys Ile Thr 515 520 525 Gln Leu Pro Ala Thr LysGly Tyr Asn Val Ser Ile Val Lys Glu Pro 530 535 540 Gly Phe Ile Gly GlyAsp Ile Gly Lys Asn Asn Gly Gln Ile Leu Gly 545 550 555 560 Lys Tyr LysVal Asn Val Glu Asp Val Ser Gln Lys Tyr Arg Phe Arg 565 570 575 Val ArgTyr Ala Thr Glu Thr Glu Gly Glu Leu Gly Ile Lys Ile Asp 580 585 590 GlyArg Thr Val Asn Leu Tyr Gln Tyr Lys Lys Thr Lys Ala Pro Gly 595 600 605Asp Pro Leu Thr Tyr Lys Ala Phe Asp Tyr Leu Ser Phe Ser Thr Pro 610 615620 Val Lys Phe Asn Asn Ala Ser Ser Thr Ile Glu Leu Phe Leu Gln Asn 625630 635 640 Lys Thr Ser Gly Thr Phe Tyr Leu Ala Gly Ile Glu Ile Ile ProVal 645 650 655 Lys Ser Asn Tyr Glu Glu Glu Leu Thr Leu Glu Glu Ala LysLys Ala 660 665 670 Val Ser Ser Leu Phe Thr Asp Ala Arg Asn Ala Leu LysIle Asp Val 675 680 685 Thr Asp Tyr Gln Ile Asp Gln Ala Ala Asn Leu ValGlu Cys Ile Ser 690 695 700 Gly Asp Leu Tyr Ala Lys Glu Lys Ile Val LeuLeu Arg Ala Val Lys 705 710 715 720 Phe Ala Lys Gln Leu Ser Gln Ser GlnAsn Leu Leu Ser Asp Pro Glu 725 730 735 Phe Asn Asn Val Asn Arg Glu AsnSer Trp Thr Ala Ser Thr Ser Val 740 745 750 Ala Ile Ile Glu Gly Asp ProLeu Tyr Lys Gly Arg Ala Val Gln Leu 755 760 765 Ser Ser Ala Arg Asp GluAsn Phe Pro Thr Tyr Leu Tyr Gln Lys Ile 770 775 780 Asp Glu Ser Thr LeuLys Pro Tyr Thr Arg Tyr Gln Leu Arg Gly Phe 785 790 795 800 Val Glu GlySer Glu Asn Leu Asp Val Tyr Leu Ile Arg Tyr Gly Ala 805 810 815 Ala HisVal Arg Met Asn Val Pro Tyr Asn Leu Glu Ile Ile Asp Thr 820 825 830 SerSer Pro Val Asn Pro Cys Glu Glu Val Asp Gly Leu Ser His Arg 835 840 845Ser Cys Asn Val Phe Asp Arg Cys Lys Gln Ser Ile Ser Val Ala Pro 850 855860 Asp Ala Asn Thr Gly Pro Asp Gln Ile Asp Gly Asp Pro His Ala Phe 865870 875 880 Ser Phe His Ile Asp Thr Gly Thr Val Asp Ser Thr Glu Asn LeuGly 885 890 895 Ile Trp Val Ala Phe Lys Ile Ser Glu Leu Asp Gly Ser AlaIle Phe 900 905 910 Gly Asn Leu Glu Leu Ile Glu Val Gly Pro Leu Ser GlyGlu Ala Leu 915 920 925 Ala Gln Val Gln Arg Lys Glu Glu Lys Trp Lys GlnVal Leu Ala Lys 930 935 940 Lys Arg Glu Thr Thr Ala Gln Thr Val Cys SerGly Glu Ala Ser Gln 945 950 955 960 Leu Thr Asn Ser Ser Gln Ile Leu LysIle Arg Asn Tyr Asp Leu Ile 965 970 975 Gln Asn Phe Arg Ile Phe Ser LeuArg Asn Thr Leu Ser Ile Lys Phe 980 985 990 Lys Ile Tyr Thr Ile Thr AsnTyr Pro Tyr Ser Arg Leu Asn Tyr Asp 995 1000 1005 Leu Phe Met Glu LeuGlu Asn Arg Ile Gln Asn Ala Ser Leu Tyr 1010 1015 1020 Met Thr Ser AsnIle Leu Gln Asn Gly Gly Phe Lys Ser Asp Val 1025 1030 1035 Thr Ser TrpGlu Thr Thr Ala Asn Ala Glu Val Gln Gln Ile Asp 1040 1045 1050 Gly AlaSer Val Leu Val Leu Ser Asn Trp Asn Ala Ser Val Ala 1055 1060 1065 GlnSer Val Asn Val Gln Asn Asp His Gly Tyr Val Leu Arg Val 1070 1075 1080Thr Ala Lys Lys Glu Gly Ile Gly Asn Gly Tyr Val Thr Ile Leu 1085 10901095 Asp Cys Ala Asn His Ile Asp Thr Leu Thr Phe Ser Ala Cys Arg 11001105 1110 Ser Asp Ser Asp Thr Ser Ser Asn Glu Leu Thr Ala Tyr Val Thr1115 1120 1125 Lys Thr Leu Glu Ile Phe Pro Asp Thr Glu Gln Ile Arg IleGlu 1130 1135 1140 Ile Gly Glu Thr Glu Gly Met Phe Tyr Val Glu Ser ValGlu Leu 1145 1150 1155 Ile Arg Met Glu Asn 1160 3 4896 DNA Bacillusthuringiensis source (1)..(4896) Bacillus thuringiensis supsp. finitimusstrain VKPM B-1161 (taxon29337 3 gatcggccga atcgggacct atcctatgaggaagttatga aaattttagg gttttataaa 60 gaacaaggtc atttagtaaa ctatacaattttactagctc tggcaagtac cggtgcaaga 120 acttcaagaa ttatgtacaa caagggttaaagacttacat tatgacggaa agcactggtt 180 aaaagttata ggtaaaggaa gtaaagtacgtgaacttttc atttctgaac atttatatga 240 gtgtatttgt gaaatgagaa gaagaagagggttccaaact gtattggacc gaggagatga 300 aagtcccttt atttgtaaat caaagagggaacttttataa ttcaaaaacg ttatcgaacc 360 aggtaacaga tatgataaaa aagaccaatttagagtttct gcagtatcgt gaaaatcctg 420 taacggcgca tacattccgt catgcttttgcaatcatggc agttgaacaa ggaaatgcag 480 atttatatca tttaatgcaa acattggggcatgaaaatat tcaaacaaca aagatttatt 540 tagaaaagca catgaaaaga aagaataatgtgggtacttc ctttgcggat atgttggttt 600 aaattttgta agatatttat ttatgtaattattatataat tacccgcata tttatggtcg 660 tttattgaac tgtgaagatt agtcatgtggcaaagttaaa aaaatagcca gtaattgttg 720 tagtttgttc tgtcgctttg ccgtacaatgaattacagac attttttata gtaacttaaa 780 taatagtcgt attttgcaaa agggtctttgtgttttcaaa ctttgcaaca gaaccatctt 840 aggtaagcca gttgatgaag gaacgtggaaaaattgaaaa acagtttagt aatctcaaag 900 ataaagggct ggaacagcca cgttggtatggaagaaatca ttatctatta catgttcagc 960 ttgtttttct gattcataac cttgcatgtttattttagtt ttgcaacacc cgcaataatt 1020 gtaacaaaac tatcaaaatc taatatactatattaaattt cagcaaaata atcaaaattt 1080 attattttta caattgaaac taaaattctaataaaaggta gtggtggg atg gca caa 1137 Met Ala Gln 1 aca tat tac aaa attgga gtt caa agt aca gaa gtt aat tct gaa tca 1185 Thr Tyr Tyr Lys Ile GlyVal Gln Ser Thr Glu Val Asn Ser Glu Ser 5 10 15 atc ttt ttt aat cca gaggtg gat agc agt gat aca gtc gct gta gta 1233 Ile Phe Phe Asn Pro Glu ValAsp Ser Ser Asp Thr Val Ala Val Val 20 25 30 35 agc gca ggg att gta gttgtg ggt act ata ctg aca gcc ttt gca tca 1281 Ser Ala Gly Ile Val Val ValGly Thr Ile Leu Thr Ala Phe Ala Ser 40 45 50 ttt gtt aat cca ggt gtg gtactt ata tca ttt gga acc ttg gct ccc 1329 Phe Val Asn Pro Gly Val Val LeuIle Ser Phe Gly Thr Leu Ala Pro 55 60 65 gtt ctt tgg cct gat cca gag gaagat cca aaa aaa att tgg tca caa 1377 Val Leu Trp Pro Asp Pro Glu Glu AspPro Lys Lys Ile Trp Ser Gln 70 75 80 ttt atg aaa cac gga gaa gac ctt ttaaat caa aca att tct aca gct 1425 Phe Met Lys His Gly Glu Asp Leu Leu AsnGln Thr Ile Ser Thr Ala 85 90 95 gta aaa gaa ata gca tta gct cat cta aatggt ttt aaa gat gta tta 1473 Val Lys Glu Ile Ala Leu Ala His Leu Asn GlyPhe Lys Asp Val Leu 100 105 110 115 acg tac tat gaa aga gca ttt aat gattgg aag aga aat cca agt gca 1521 Thr Tyr Tyr Glu Arg Ala Phe Asn Asp TrpLys Arg Asn Pro Ser Ala 120 125 130 aat act gcc aga ttg gta tca cag agattt gaa aac gct cat ttc aat 1569 Asn Thr Ala Arg Leu Val Ser Gln Arg PheGlu Asn Ala His Phe Asn 135 140 145 ttt gta agc aat atg cca caa ctc caactt ccc acg tat gac aca tta 1617 Phe Val Ser Asn Met Pro Gln Leu Gln LeuPro Thr Tyr Asp Thr Leu 150 155 160 tta tta agt tgc tat aca gaa gct gcaaat tta cat ttg aat tta tta 1665 Leu Leu Ser Cys Tyr Thr Glu Ala Ala AsnLeu His Leu Asn Leu Leu 165 170 175 cat caa ggt gta caa ttc gcg gat caatgg aat gca gat caa cca cat 1713 His Gln Gly Val Gln Phe Ala Asp Gln TrpAsn Ala Asp Gln Pro His 180 185 190 195 tca cca atg ttg aag tca tca ggtact tat tat gac gag cta ttg gta 1761 Ser Pro Met Leu Lys Ser Ser Gly ThrTyr Tyr Asp Glu Leu Leu Val 200 205 210 tat att gaa aag tat att aat tattgc acc aag aca tac cat aaa gga 1809 Tyr Ile Glu Lys Tyr Ile Asn Tyr CysThr Lys Thr Tyr His Lys Gly 215 220 225 ttg aat cac ctt aaa gaa tca gaaaaa atc aca tgg gat gct tat aac 1857 Leu Asn His Leu Lys Glu Ser Glu LysIle Thr Trp Asp Ala Tyr Asn 230 235 240 aca tat cgt cga gaa atg acc ttaatt gta ttg gat ctt gtc gca act 1905 Thr Tyr Arg Arg Glu Met Thr Leu IleVal Leu Asp Leu Val Ala Thr 245 250 255 ttt cct ttt tat gat ata cgt cgtttt cca aga gga gta gaa cta gaa 1953 Phe Pro Phe Tyr Asp Ile Arg Arg PhePro Arg Gly Val Glu Leu Glu 260 265 270 275 tta aca aga gag gtt tat acaagt tta gat cat tta aca cga cca cca 2001 Leu Thr Arg Glu Val Tyr Thr SerLeu Asp His Leu Thr Arg Pro Pro 280 285 290 ggg cta ttt act tgg ctg tcagat att gag tta tac acg gag agt gtg 2049 Gly Leu Phe Thr Trp Leu Ser AspIle Glu Leu Tyr Thr Glu Ser Val 295 300 305 gca gaa ggc gat tat tta tcaggt att cga gag tct aaa tat tat act 2097 Ala Glu Gly Asp Tyr Leu Ser GlyIle Arg Glu Ser Lys Tyr Tyr Thr 310 315 320 ggt aat caa ttt ttt acg atgaaa aat att tat ggt aat aca aat aga 2145 Gly Asn Gln Phe Phe Thr Met LysAsn Ile Tyr Gly Asn Thr Asn Arg 325 330 335 tta agt aag cag ctc att acatta tta cca ggc gaa ttt atg act cac 2193 Leu Ser Lys Gln Leu Ile Thr LeuLeu Pro Gly Glu Phe Met Thr His 340 345 350 355 tta agc ata aac cgt cctttt caa aca ata gct ggt ata aat aag tta 2241 Leu Ser Ile Asn Arg Pro PheGln Thr Ile Ala Gly Ile Asn Lys Leu 360 365 370 tac agt tta att caa aaaatc gta ttc aca act ttt aaa aac gat aat 2289 Tyr Ser Leu Ile Gln Lys IleVal Phe Thr Thr Phe Lys Asn Asp Asn 375 380 385 gaa tat caa aaa aat tttaat gtg aat aat caa aat gaa cct caa gaa 2337 Glu Tyr Gln Lys Asn Phe AsnVal Asn Asn Gln Asn Glu Pro Gln Glu 390 395 400 act aca aac tat cct aatgat tat ggt ggt tca aac agc caa aaa ttc 2385 Thr Thr Asn Tyr Pro Asn AspTyr Gly Gly Ser Asn Ser Gln Lys Phe 405 410 415 aaa cat aat tta tct catttt cca tta atc atc cac aag tta gag ttt 2433 Lys His Asn Leu Ser His PhePro Leu Ile Ile His Lys Leu Glu Phe 420 425 430 435 gct gag tat ttt cactct ata ttt gca tta ggt tgg aca cac aat agt 2481 Ala Glu Tyr Phe His SerIle Phe Ala Leu Gly Trp Thr His Asn Ser 440 445 450 gta aac tcc caa aattta ata tca gaa agt gtg agt aca caa atc cca 2529 Val Asn Ser Gln Asn LeuIle Ser Glu Ser Val Ser Thr Gln Ile Pro 455 460 465 ttg gta aaa gct tacgaa gtt act aac aat tca gtt ata aga gga cca 2577 Leu Val Lys Ala Tyr GluVal Thr Asn Asn Ser Val Ile Arg Gly Pro 470 475 480 ggt ttt aca ggt ggagat tta ata gaa ctt cgt gat aaa tgt tct att 2625 Gly Phe Thr Gly Gly AspLeu Ile Glu Leu Arg Asp Lys Cys Ser Ile 485 490 495 aaa tgt aaa gct agttct tta aaa aaa tac gct ata agt cta ttt tat 2673 Lys Cys Lys Ala Ser SerLeu Lys Lys Tyr Ala Ile Ser Leu Phe Tyr 500 505 510 515 gct gca aat aacgca ata gct gta tca ata gac gta ggt gat tcc gga 2721 Ala Ala Asn Asn AlaIle Ala Val Ser Ile Asp Val Gly Asp Ser Gly 520 525 530 gca gga gtt ctattg caa cct acc ttt tct aga aaa ggg aac aat aat 2769 Ala Gly Val Leu LeuGln Pro Thr Phe Ser Arg Lys Gly Asn Asn Asn 535 540 545 ttt aca att caagac ctt aac tat aag gat ttt caa tat cat aca ctt 2817 Phe Thr Ile Gln AspLeu Asn Tyr Lys Asp Phe Gln Tyr His Thr Leu 550 555 560 tta gtt gat attgaa tta ccc gaa agt gaa gaa att cat atc cat ttg 2865 Leu Val Asp Ile GluLeu Pro Glu Ser Glu Glu Ile His Ile His Leu 565 570 575 aag cga gag gatgat tat gag gag gga gtg att ctt tta att gat aaa 2913 Lys Arg Glu Asp AspTyr Glu Glu Gly Val Ile Leu Leu Ile Asp Lys 580 585 590 595 tta gag ttcaaa cct ata gat gaa aat tat act aat gaa atg aat tta 2961 Leu Glu Phe LysPro Ile Asp Glu Asn Tyr Thr Asn Glu Met Asn Leu 600 605 610 gag aag gcaaag aaa gca gtg aat gta tta ttt ata aac gca aca aac 3009 Glu Lys Ala LysLys Ala Val Asn Val Leu Phe Ile Asn Ala Thr Asn 615 620 625 gct ttg aaaatg gac gta act gat tat cac att gat caa gtg gca aac 3057 Ala Leu Lys MetAsp Val Thr Asp Tyr His Ile Asp Gln Val Ala Asn 630 635 640 tta gta gaatgt ata tcg gac gac cta tat gca aag gaa aaa att aaa 3105 Leu Val Glu CysIle Ser Asp Asp Leu Tyr Ala Lys Glu Lys Ile Lys 645 650 655 ttt act ccatgt att aaa ttc gcg aaa caa ttg agt caa gca cga aat 3153 Phe Thr Pro CysIle Lys Phe Ala Lys Gln Leu Ser Gln Ala Arg Asn 660 665 670 675 cta ttatcc gat ccg aat ttt aac aat cta aac gct gaa aat agt tgg 3201 Leu Leu SerAsp Pro Asn Phe Asn Asn Leu Asn Ala Glu Asn Ser Trp 680 685 690 aca gcaaat aca ggt gtc aca atc att gaa gga gac cca ttg tat aaa 3249 Thr Ala AsnThr Gly Val Thr Ile Ile Glu Gly Asp Pro Leu Tyr Lys 695 700 705 ggg cgtgct att caa tta tca gcc gcg agg gat gaa aac ttt cca act 3297 Gly Arg AlaIle Gln Leu Ser Ala Ala Arg Asp Glu Asn Phe Pro Thr 710 715 720 tat ctgtac caa aaa ata gat gaa tcc tta tta aaa cct tat aca cgt 3345 Tyr Leu TyrGln Lys Ile Asp Glu Ser Leu Leu Lys Pro Tyr Thr Arg 725 730 735 tat caacta aga gga ttt gta gaa ggt agt caa gat tta gaa ctc gat 3393 Tyr Gln LeuArg Gly Phe Val Glu Gly Ser Gln Asp Leu Glu Leu Asp 740 745 750 755 ttggta cgc tac ggg gca aca gac att gta atg aat gtg ccc ggc gac 3441 Leu ValArg Tyr Gly Ala Thr Asp Ile Val Met Asn Val Pro Gly Asp 760 765 770 cttgaa atc ctc agt tac tct gcc cct atc aat cct tgt gag gaa ata 3489 Leu GluIle Leu Ser Tyr Ser Ala Pro Ile Asn Pro Cys Glu Glu Ile 775 780 785 gaaaca cgc tta gat act act tgt ggt gcg ctt gat cgt tgt aag caa 3537 Glu ThrArg Leu Asp Thr Thr Cys Gly Ala Leu Asp Arg Cys Lys Gln 790 795 800 tccaat tat gta aat tca gct gca gat gta agg cct gat caa gtg aat 3585 Ser AsnTyr Val Asn Ser Ala Ala Asp Val Arg Pro Asp Gln Val Asn 805 810 815 ggagat cca cac gca ttt tca ttc cat att gat aca ggt act acg gat 3633 Gly AspPro His Ala Phe Ser Phe His Ile Asp Thr Gly Thr Thr Asp 820 825 830 835aat aat aga aat tta ggg att tgg att att ttt aaa att gcc aca cca 3681 AsnAsn Arg Asn Leu Gly Ile Trp Ile Ile Phe Lys Ile Ala Thr Pro 840 845 850gac ggc tat gca act ttc ggt aat cta gaa ttg ata gaa ttg gga cca 3729 AspGly Tyr Ala Thr Phe Gly Asn Leu Glu Leu Ile Glu Leu Gly Pro 855 860 865tta tct gga gaa gcg tta gca caa gta caa cgg aaa gaa caa aaa tgg 3777 LeuSer Gly Glu Ala Leu Ala Gln Val Gln Arg Lys Glu Gln Lys Trp 870 875 880gga aaa aac aca acc caa aaa agg gaa gaa gct gca aaa tta tat gca 3825 GlyLys Asn Thr Thr Gln Lys Arg Glu Glu Ala Ala Lys Leu Tyr Ala 885 890 895gct gca aag caa aca att aat caa tta ttc gcc gat tca caa ggt aca 3873 AlaAla Lys Gln Thr Ile Asn Gln Leu Phe Ala Asp Ser Gln Gly Thr 900 905 910915 aaa tta aga ttt gat aca gaa ttc tcc aat att tta tcg gca gat aaa 3921Lys Leu Arg Phe Asp Thr Glu Phe Ser Asn Ile Leu Ser Ala Asp Lys 920 925930 ctt gtc tat aaa att cga gat gta tat agt gaa gtt tta tct gtt atc 3969Leu Val Tyr Lys Ile Arg Asp Val Tyr Ser Glu Val Leu Ser Val Ile 935 940945 cca gga tta aat tat gat tta ttt atg gaa ctt gaa aat aga att cag 4017Pro Gly Leu Asn Tyr Asp Leu Phe Met Glu Leu Glu Asn Arg Ile Gln 950 955960 aat gca att gat tta tat gac gct cgc aat acc gtg aca aat ggg gag 4065Asn Ala Ile Asp Leu Tyr Asp Ala Arg Asn Thr Val Thr Asn Gly Glu 965 970975 ttt aga aat ggt ttg gcg aat tgg atg gct tca tca aat aca gaa gta 4113Phe Arg Asn Gly Leu Ala Asn Trp Met Ala Ser Ser Asn Thr Glu Val 980 985990 995 agg caa atc cag gca cat ccg tgt tgg tac tct cta ggc tgg aat 4158Arg Gln Ile Gln Ala His Pro Cys Trp Tyr Ser Leu Gly Trp Asn 1000 10051010 gcg cag gtt gca caa tct cta aat gtg aaa cct gat cat ggg tat 4203Ala Gln Val Ala Gln Ser Leu Asn Val Lys Pro Asp His Gly Tyr 1015 10201025 gta tta cgt gta aca gca aaa aaa gaa gga att ggt aat ggc tat 4248Val Leu Arg Val Thr Ala Lys Lys Glu Gly Ile Gly Asn Gly Tyr 1030 10351040 gtg aca atc ctt gac tgt gca aat cat att gat acg ttg aca ttt 4293Val Thr Ile Leu Asp Cys Ala Asn His Ile Asp Thr Leu Thr Phe 1045 10501055 agt tct tgt gat tca ggt ttc act act tct tct aat gaa tta gca 4338Ser Ser Cys Asp Ser Gly Phe Thr Thr Ser Ser Asn Glu Leu Ala 1060 10651070 gcc tat gtt aca aaa acg tta gaa att ttc cca gat acc gat caa 4383Ala Tyr Val Thr Lys Thr Leu Glu Ile Phe Pro Asp Thr Asp Gln 1075 10801085 att cgc att gaa atc ggc gaa acc cga agt acg ttt tat gta gaa 4428Ile Arg Ile Glu Ile Gly Glu Thr Arg Ser Thr Phe Tyr Val Glu 1090 10951100 agt gtg gac cta att cga atg gag gat tga ttggagaggt ttatcatata 4478Ser Val Asp Leu Ile Arg Met Glu Asp 1105 ttaaaaataa aggtgaggtctcctctatga ggacctcgct tttgttttaa tatgaacgtt 4538 ctagtaagac tcgaagtcactataggtatt aagagattac tagaatataa aaggacaact 4598 ttccattagg agaatattacttttatgttt ttgtcctgat ttttcttgta aaatcaaaat 4658 tcttacagat ccctttaatttctcaacaac ttgtttttgg attcctttct catactttgg 4718 caatcacatt taacgcggacatggtaatta atttgcagac cgaattaacg ttttgtgtcc 4778 actcctgata catacccttcttcaatgttt taatgattct aattcttgta tagtcaatgg 4838 ttcattactt agatttgtcatttcaatttc ccaaattttt tagaatttct ttttgatc 4896 4 1109 PRT Bacillusthuringiensis 4 Met Ala Gln Thr Tyr Tyr Lys Ile Gly Val Gln Ser Thr GluVal Asn 1 5 10 15 Ser Glu Ser Ile Phe Phe Asn Pro Glu Val Asp Ser SerAsp Thr Val 20 25 30 Ala Val Val Ser Ala Gly Ile Val Val Val Gly Thr IleLeu Thr Ala 35 40 45 Phe Ala Ser Phe Val Asn Pro Gly Val Val Leu Ile SerPhe Gly Thr 50 55 60 Leu Ala Pro Val Leu Trp Pro Asp Pro Glu Glu Asp ProLys Lys Ile 65 70 75 80 Trp Ser Gln Phe Met Lys His Gly Glu Asp Leu LeuAsn Gln Thr Ile 85 90 95 Ser Thr Ala Val Lys Glu Ile Ala Leu Ala His LeuAsn Gly Phe Lys 100 105 110 Asp Val Leu Thr Tyr Tyr Glu Arg Ala Phe AsnAsp Trp Lys Arg Asn 115 120 125 Pro Ser Ala Asn Thr Ala Arg Leu Val SerGln Arg Phe Glu Asn Ala 130 135 140 His Phe Asn Phe Val Ser Asn Met ProGln Leu Gln Leu Pro Thr Tyr 145 150 155 160 Asp Thr Leu Leu Leu Ser CysTyr Thr Glu Ala Ala Asn Leu His Leu 165 170 175 Asn Leu Leu His Gln GlyVal Gln Phe Ala Asp Gln Trp Asn Ala Asp 180 185 190 Gln Pro His Ser ProMet Leu Lys Ser Ser Gly Thr Tyr Tyr Asp Glu 195 200 205 Leu Leu Val TyrIle Glu Lys Tyr Ile Asn Tyr Cys Thr Lys Thr Tyr 210 215 220 His Lys GlyLeu Asn His Leu Lys Glu Ser Glu Lys Ile Thr Trp Asp 225 230 235 240 AlaTyr Asn Thr Tyr Arg Arg Glu Met Thr Leu Ile Val Leu Asp Leu 245 250 255Val Ala Thr Phe Pro Phe Tyr Asp Ile Arg Arg Phe Pro Arg Gly Val 260 265270 Glu Leu Glu Leu Thr Arg Glu Val Tyr Thr Ser Leu Asp His Leu Thr 275280 285 Arg Pro Pro Gly Leu Phe Thr Trp Leu Ser Asp Ile Glu Leu Tyr Thr290 295 300 Glu Ser Val Ala Glu Gly Asp Tyr Leu Ser Gly Ile Arg Glu SerLys 305 310 315 320 Tyr Tyr Thr Gly Asn Gln Phe Phe Thr Met Lys Asn IleTyr Gly Asn 325 330 335 Thr Asn Arg Leu Ser Lys Gln Leu Ile Thr Leu LeuPro Gly Glu Phe 340 345 350 Met Thr His Leu Ser Ile Asn Arg Pro Phe GlnThr Ile Ala Gly Ile 355 360 365 Asn Lys Leu Tyr Ser Leu Ile Gln Lys IleVal Phe Thr Thr Phe Lys 370 375 380 Asn Asp Asn Glu Tyr Gln Lys Asn PheAsn Val Asn Asn Gln Asn Glu 385 390 395 400 Pro Gln Glu Thr Thr Asn TyrPro Asn Asp Tyr Gly Gly Ser Asn Ser 405 410 415 Gln Lys Phe Lys His AsnLeu Ser His Phe Pro Leu Ile Ile His Lys 420 425 430 Leu Glu Phe Ala GluTyr Phe His Ser Ile Phe Ala Leu Gly Trp Thr 435 440 445 His Asn Ser ValAsn Ser Gln Asn Leu Ile Ser Glu Ser Val Ser Thr 450 455 460 Gln Ile ProLeu Val Lys Ala Tyr Glu Val Thr Asn Asn Ser Val Ile 465 470 475 480 ArgGly Pro Gly Phe Thr Gly Gly Asp Leu Ile Glu Leu Arg Asp Lys 485 490 495Cys Ser Ile Lys Cys Lys Ala Ser Ser Leu Lys Lys Tyr Ala Ile Ser 500 505510 Leu Phe Tyr Ala Ala Asn Asn Ala Ile Ala Val Ser Ile Asp Val Gly 515520 525 Asp Ser Gly Ala Gly Val Leu Leu Gln Pro Thr Phe Ser Arg Lys Gly530 535 540 Asn Asn Asn Phe Thr Ile Gln Asp Leu Asn Tyr Lys Asp Phe GlnTyr 545 550 555 560 His Thr Leu Leu Val Asp Ile Glu Leu Pro Glu Ser GluGlu Ile His 565 570 575 Ile His Leu Lys Arg Glu Asp Asp Tyr Glu Glu GlyVal Ile Leu Leu 580 585 590 Ile Asp Lys Leu Glu Phe Lys Pro Ile Asp GluAsn Tyr Thr Asn Glu 595 600 605 Met Asn Leu Glu Lys Ala Lys Lys Ala ValAsn Val Leu Phe Ile Asn 610 615 620 Ala Thr Asn Ala Leu Lys Met Asp ValThr Asp Tyr His Ile Asp Gln 625 630 635 640 Val Ala Asn Leu Val Glu CysIle Ser Asp Asp Leu Tyr Ala Lys Glu 645 650 655 Lys Ile Lys Phe Thr ProCys Ile Lys Phe Ala Lys Gln Leu Ser Gln 660 665 670 Ala Arg Asn Leu LeuSer Asp Pro Asn Phe Asn Asn Leu Asn Ala Glu 675 680 685 Asn Ser Trp ThrAla Asn Thr Gly Val Thr Ile Ile Glu Gly Asp Pro 690 695 700 Leu Tyr LysGly Arg Ala Ile Gln Leu Ser Ala Ala Arg Asp Glu Asn 705 710 715 720 PhePro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Leu Leu Lys Pro 725 730 735Tyr Thr Arg Tyr Gln Leu Arg Gly Phe Val Glu Gly Ser Gln Asp Leu 740 745750 Glu Leu Asp Leu Val Arg Tyr Gly Ala Thr Asp Ile Val Met Asn Val 755760 765 Pro Gly Asp Leu Glu Ile Leu Ser Tyr Ser Ala Pro Ile Asn Pro Cys770 775 780 Glu Glu Ile Glu Thr Arg Leu Asp Thr Thr Cys Gly Ala Leu AspArg 785 790 795 800 Cys Lys Gln Ser Asn Tyr Val Asn Ser Ala Ala Asp ValArg Pro Asp 805 810 815 Gln Val Asn Gly Asp Pro His Ala Phe Ser Phe HisIle Asp Thr Gly 820 825 830 Thr Thr Asp Asn Asn Arg Asn Leu Gly Ile TrpIle Ile Phe Lys Ile 835 840 845 Ala Thr Pro Asp Gly Tyr Ala Thr Phe GlyAsn Leu Glu Leu Ile Glu 850 855 860 Leu Gly Pro Leu Ser Gly Glu Ala LeuAla Gln Val Gln Arg Lys Glu 865 870 875 880 Gln Lys Trp Gly Lys Asn ThrThr Gln Lys Arg Glu Glu Ala Ala Lys 885 890 895 Leu Tyr Ala Ala Ala LysGln Thr Ile Asn Gln Leu Phe Ala Asp Ser 900 905 910 Gln Gly Thr Lys LeuArg Phe Asp Thr Glu Phe Ser Asn Ile Leu Ser 915 920 925 Ala Asp Lys LeuVal Tyr Lys Ile Arg Asp Val Tyr Ser Glu Val Leu 930 935 940 Ser Val IlePro Gly Leu Asn Tyr Asp Leu Phe Met Glu Leu Glu Asn 945 950 955 960 ArgIle Gln Asn Ala Ile Asp Leu Tyr Asp Ala Arg Asn Thr Val Thr 965 970 975Asn Gly Glu Phe Arg Asn Gly Leu Ala Asn Trp Met Ala Ser Ser Asn 980 985990 Thr Glu Val Arg Gln Ile Gln Ala His Pro Cys Trp Tyr Ser Leu Gly 9951000 1005 Trp Asn Ala Gln Val Ala Gln Ser Leu Asn Val Lys Pro Asp His1010 1015 1020 Gly Tyr Val Leu Arg Val Thr Ala Lys Lys Glu Gly Ile GlyAsn 1025 1030 1035 Gly Tyr Val Thr Ile Leu Asp Cys Ala Asn His Ile AspThr Leu 1040 1045 1050 Thr Phe Ser Ser Cys Asp Ser Gly Phe Thr Thr SerSer Asn Glu 1055 1060 1065 Leu Ala Ala Tyr Val Thr Lys Thr Leu Glu IlePhe Pro Asp Thr 1070 1075 1080 Asp Gln Ile Arg Ile Glu Ile Gly Glu ThrArg Ser Thr Phe Tyr 1085 1090 1095 Val Glu Ser Val Asp Leu Ile Arg MetGlu Asp 1100 1105 5 27 DNA Artificial sequence putative vegetativepromoter sequence (1)..(27) n = a, t, c, or g 5 ttgcaannnn nnnnnnnnnnntaagcc 27

What is claimed is:
 1. An isolated nucleic acid molecule, comprising:(a) a nucleotide sequence that encodes a polypeptide at least 90%identical to SEQ ID NO: 2 or SEQ ID NO: 4; (b) a nucleotide sequencethat encodes SEQ ID NO: 2 or SEQ ID NO: 4; (c) nucleotides 897-4388 ofSEQ ID NO: 1 or nucleotides 1129-4458 of SEQ ID NO: 3; (d) a consecutive20 base pair nucleotide portion identical in sequence to a consecutive20 base pair portion of nucleotides 897-4388 of SEQ ID NO: 1 or aconsecutive 20 base pair portion of nucleotides 1129-4458 of SEQ ID NO:3; or (e) a nucleotide sequence whose complement hybridizes understringent hybridization and wash conditions to nucleotides 897-4388 ofSEQ ID NO: 1 or nucleotides 1129-4458 of SEQ ID NO: 3; wherein saidnucleic acid molecule encodes a toxin that is active against insects. 2.An isolated nucleic acid molecule according to claim 1, wherein saidnucleotide sequence encodes a polypeptide at least 90% identical to SEQID NO:
 2. 3. An isolated nucleic acid molecule according to claim 1,wherein said nucleotide sequence encodes a polypeptide at least 90%identical to SEQ ID NO:
 4. 4. An isolated nucleic acid moleculeaccording to claim 1, wherein said nucleotide sequence encodes the aminoacid sequence set forth as SEQ ID NO:
 2. 5. An isolated nucleic acidmolecule according to claim 1, wherein said nucleotide sequence encodesthe amino acid sequence set forth as SEQ ID NO:
 4. 6. An isolatednucleic acid molecule according to claim 1, comprising nucleotides897-4388 of SEQ ID NO:
 1. 7. An isolated nucleic acid molecule accordingto claim 1, comprising nucleotides 1129-4458 of SEQ ID NO:
 3. 8. Anisolated nucleic acid molecule according to claim 1, wherein saidnucleic acid molecule comprises a 20 base pair nucleotide portionidentical in sequence to a consecutive 20 base pair portion ofnucleotides 897-4388 of SEQ ID NO:
 1. 9. An isolated nucleic acidmolecule according to claim 1, wherein said nucleic acid moleculecomprises a 20 base pair nucleotide portion identical in sequence to aconsecutive 20 base pair portion of nucleotides 1129-4458 of SEQ ID NO:3.
 10. An isolated nucleic acid molecule according to claim 1,comprising a nucleotide sequence whose complement hybridizes understringent hybridization and wash conditions to nucleotides 897-4388 ofSEQ ID NO:
 1. 11. An isolated nucleic acid molecule according to claim1, comprising a nucleotide sequence whose complement hybridizes understringent hybridization and wash conditions to nucleotides 1129-4458 ofSEQ ID NO:
 3. 12. A chimeric construct comprising a heterologouspromoter sequence operatively linked to the nucleic acid molecule ofclaim
 1. 13. A recombinant vector comprising the chimeric construct ofclaim
 12. 14. A transgenic host cell comprising the chimeric constructof claim
 12. 15. A transgenic host cell according to claim 14, which isa transgenic bacterial cell.
 16. A transgenic host cell according toclaim 14, which is a transgenic plant cell.
 17. A transgenic plantcomprising the transgenic plant cell of claim
 16. 18. A transgenic plantaccording to claim 17, which is maize.
 19. Seed from the transgenicplant of claim
 17. 20. A toxin produced by expression of a DNA moleculeaccording to claim
 1. 21. A toxin according to claim 20, wherein saidtoxin comprises the amino acid sequence set forth as SEQ ID NO:
 2. 22. Atoxin according to claim 20, wherein said toxin comprises the amino acidsequence set forth as SEQ ID NO:
 4. 23. A toxin according to claim 20,wherein said toxin comprises an amino acid sequence at least 90%identical to SEQ ID NO:
 2. 24. A toxin according to claim 20, whereinsaid toxin comprises an amino acid sequence at least 90% identical toSEQ ID NO:
 4. 25. A composition comprising an insecticidally effectiveamount of a toxin according to claim
 20. 26. A method of producing atoxin that is active against insects, comprising: (a) obtaining atransgenic host cell according to claim 14; and (b) expressing thenucleic acid molecule in said transgenic cell, which results in thetoxin that is active against insects.
 25. A method of producing aninsect-resistant plant, comprising introducing a nucleic acid moleculeaccording to claim 1 into said plant, wherein said nucleic acid moleculeis expressible in said plant in an effective amount to control aninsect.
 26. A method of controlling an insect comprising delivering tothe insect an effective amount of a toxin according to claim
 20. 27. Amethod for mutagenizing a nucleic acid molecule according to claim 1,wherein the nucleic acid molecule has been cleaved into population ofdouble-stranded random fragments of a desired size, comprising: (a)adding to the population of double-stranded random fragments one or moresingle- or double-stranded oligonucleotides, wherein saidoligonucleotides each comprise an area of identity and an area ofheterology to a double-stranded template polynucleotide; (b) denaturingthe resultant mixture of double-stranded random fragments andoligonucleotides into single-stranded fragments; (c) incubating theresultant population of single-stranded fragments with a polymeraseunder conditions which result in the annealing of said single-strandedfragments at said areas of identity to form pairs of annealed fragments,said areas of identity being sufficient for one member of a pair toprime replication of the other, thereby forming a mutagenizeddouble-stranded polynucleotide; and (d) repeating the second and thirdsteps for at least two further cycles, wherein the resultant mixture inthe second step of a further cycle includes the mutagenizeddouble-stranded polynucleotide from the third step of the previouscycle, and wherein the further cycle forms a further mutagenizeddouble-stranded polynucleotide.